Cyclosporin derivatives

ABSTRACT

The present invention relates to novel cyclosporin derivatives that do not cross the cellular membrane. The compounds according to the invention are used in medicine, more particularly in the treatment/diagnosis of acute and chronic inflammatory diseases, viral infections, cancer, degenerative muscle diseases, neurodegenerative diseases and damage that is associated with calcium homeostasis impairment. The novel cyclosporin derivatives additionally have no immunosuppressive effect.

The present invention is directed to compounds of formula I shown belowand tracer-coupled compounds, which are both cyclosporin derivatives.The compounds according to the present invention show noimmunosuppressive effect and are used in medicine, in particular in thetreatment of diseases like viral infections, inflammatory diseases,cancer, degenerative muscles diseases, neurodegenerative diseases anddamages that are associated with impairment of calcium homeostasis.Furthermore, the present invention is directed to a pharmaceuticalcomposition containing a compound according to the present invention andoptionally different additives. The present invention is furthermoredirected to a method of accumulation of active agents in anextracellular space of a multi-cellular object. A further objective ofthe present invention is the use of various cyclosporin derivatives forthe preparation of compounds according to the present invention.

Biologically active molecules, so-called “active agents”, which are ingeneral pharmaceutically active agents, have their effect mostly insideas well as outside of biological cells. Thereby, the problem especiallyemerged so far is that active agents, which should have their effectsonly inside the cell are causing unwanted side effects in theextracellular space already prior to their transfer through the cellmembrane. At this arises the additional problem that one and the sameactive agent can have different effects inside and outside of the cell.The actual effect comprises therefore two components—the desiredintracellular effect and the undesired extracellular effect. To achievethe intracellular effect, often also the extracellular (side) effectshave necessarily to be accepted—since the transport to the cell includesnormally crossing an extracellular space.

Equally, only the extracellular effect can be desired and theintracellular effect can be undesired. Especially in medicine, variousactive agents are known that not only do not cause the desired effect inthe cell, but are even toxic or cause an otherwise harmful effect. Itcomes along that a far higher dose than is actually needed has to beapplied for achieving a specific, extracellular effect to compensate forthe “loss” of active agents migrated into the cell.

Active agents can affect molecules or structures extracellularly and/orintracellularly. Such biological molecules could for example be enzymes,inhibitors, activators or receptors. “Structures” for example can beunderstood as extracellular matrix, consisting of the entirety ofmacromolecules, which are located outside of the plasma membrane ofcells in tissues and organs.

Various publications show that cyclosporins can have intracellular aswell as extracellular effects. Cyclosporins can bind intracellularly aswell as extracellularly to proteins, like cyclophilins. These proteinsare comprehensively described in the scientific and patent literature,such as for example in WO 2010/012073. Cyclosporins and theirderivatives have a variety of effects which may be used therapeutically.Thus, they may influence neuroprotection/neurogeneration, chaperonactivity, HIV-infection, cancer or Alzheimer's disease. An example for adesired effect of these compounds is the PPlase-inhibition. Although,the PPlase(peptidyl-prolyl-isomerase)-inhibitors could differentiatebetween the individual PPlase-families ((Nature Chemical Biology.3(10):619-29, 2007; Cellular & Molecular Life Sciences. 63(24):2889-900, 2006; Current Topics in Medicinal Chemistry.3(12):1315-47, 2003; Advances in Protein Chemistry. 59:243-82, 2001)they have often similar inhibitive strength in view of sequence-similarfamily members. As PPlases within one family can influence differentbiochemical reactions, the diagnostic or pharmacological effect ofapplied active agents depends directly on the concentration achieved indifferent distribution spaces. Thus, for example, some of thesePPlase-inhibitors (e.g. Biopolymers 84(2006)125-146; Chemical &Pharmaceutical Bulletin. 54 (3):372-376, 2006; Chemistry & Biology. 10(1):15-24, 2003; Nucleic Acids Research. 29(3):767-773, 2001), such asthe therapeutically used cyclosporin A, are only slightly soluble inwater (DE 19859910).

Cyclosporin A originally isolated from the fungi Tolypocladium inflatumis for example used in medical applications for suppression of immuneresponses. Cyclosporins, in terms of this invention, are cyclic peptidesof 11 amino acids (undecapeptides). All cyclosporins A to I, as well astheir derivatives as described in Helvetica Chimica Acta 65 (1982),1655-1677 and all cyclosporins K to Z as well as their derivatives asdescribed in Chimica Acta 70 (1987), 13-36 are included. Thecyclosporins may according to the invention also—as described in WO99/18120—be deuterated.

Cyclosporin A (also Ciclosporin) is a cyclic oligopeptide, which hasintracellularly a calcineurin-inhibiting effect. A cyclosporin,effective as such, is characterized by a selective and reversiblemechanism of immunosuppression. It blocks selectively the activation ofT-lymphocytes by production of certain cytokines, which participate inthe regulation of these T-cells. Thereby, particularly, the synthesis ofinterleukin-2 is inhibited causing in parallel that the proliferation ofcytotoxic T-lymphocytes, which for example are responsible for rejectionof foreign tissue, is suppressed. The cyclosporin takes effectintracellularly by binding to the so called cyclophilins orimmunophilins, which belong to the family of cyclosporin-bindingproteins. According to the present invention, it is preferred that thecompounds of the invention do not have this intracellular effect.

Inhibitors of cyclophilins have a very wide therapeutic range, such as,for example the treatment of diseases of the respiratory tract, likee.g. asthma, chronic obstructive pulmonary disease (COPD), pneumonia oremphysema (Expert Opinion on Investigational Drugs 12(2003)647-653,Biodrugs 8(1997) 205-215, American Journal of Respiratory Cell &Molecular Biology 20(1999)481-492), metabolic diseases, like diabetes(Transplantation Proceedings 37(2005)1857-1860, Molecular Pharmacology60(2001)873-879), inflammatory diseases of the digestive tract (BoneMarrow Transplantation 26(2000):545-551, Pharmaceutical Research20(2003)910-917), disorders of the immune system (Immunology Letters84(2002)137-143, Acta Biochimica Polonica 49(2002)233-247) inflammations(Journal of Periodontal Research 42(2007)580-588, Journal of Neurology,Neurosurgery & Psychiatry 76(2005)1115-1120, Transplant Immunology12(2004):151-157), cardiovascular diseases (Journal of Hypertension17(1999)1707-1713, Drug & Chemical Toxicology 21(1998)27-34),neurological diseases (Annals of Vascular Surgery. 20(2006) 243-249),diseases that are associated with dysfunction of angiogenesis (BloodPurification. 25 (2007) 466-472, International Angiology 24 (2005)372-379, Nefrologia. 23(2003):44-48), for suppression of the immuneresponse in case of organ transplantation (Bone Marrow Transplantation.38 (2006) 169-174), Biodrugs. 14 (2000) 185-193, ClinicalImmunotherapeutics. 5 (1996) 351-373) and of autoimmune diseases(Immunology & Immunopathology. 82(3):197-202, 1997), arthritic diseases(British Journal of Rheumatology. 36(1997)808-811, Biodrugs. 7 (1997)376-385), dermatitis (Veterinary Dermatology 17 (2006) 3-16), psoriasis(Journal of Dermatological Treatment 16(2005)258-277, Hautarzt 44 (1993)353-360), in allergies (Cornea 27 (2008) 625, Journal of Small AnimalPractice 47(2006)434-438, Clinical & Experimental Ophthalmology34(2006)347-353), for multiple sclerosis (Immunopharmacology &Immunotoxicology 21(1999)527-549, Journal of Neuroimaging 7(1997)1-7),diseases caused by ischemia such as e.g. cardiac infarction (Annals ofThoracic Surgery 86(2008)1286-1292, Acta Anaesthesiologica Scandinavica51(2007)+909-913), infarction of the pancreas (Pancreas 32 (2006)145-151) or stroke (Annals of Vascular Surgery 20(2006)243-249,Neurological Research 27(2005)827-834), kidney diseases such as e.g.glomerulonephritis (Nephrology Dialysis Transplantation 19(2004)3207,Nephron 91(2002)509-511), tumors (Journal of Investigative Dermatology128(2008)2467-2473, Endocrinology 148(2007)4716-4726), for multiplemyeloma (Leukemia 12 (1998) 505-509, Leukemia & Lymphoma16(1994)167-170), for acute or chronic leukemia (Cancer Chemotherapy &Pharmacology 52 (2003) 449-452, Cancer 97 (2003) 1481-1487), muscledegeneration (Neuroscience Research Communications 31(2002)85-92,cachexia (International Journal of Cardiology 85(2002)173-183, Drugs 58(1999) 953-963, 1999), Reiter's syndrome (Rheumatology 40(2001)945-947),diseases of bone resorption (European Journal of Pharmacology564(2007)226-231, Biochemical & Biophysical Research Communications254(1999)248-252), in Alzheimer's disease (Biochemical & BiophysicalResearch Communications 248 (1998) 168-173, Chinese Medical Journal 115(2002) 884-887), malaria (Molecular & Biochemical Parasitology99(1999)167-181), septic and toxic shock syndrome (Journal ofPharmacology & Experimental Therapeutics 311(2004)1256-1263), myalgia(British Journal of Dermatology 147(2002)606-607), in viral infection(Expert Opinion on Emerging Drugs 13 (2008) 393-416) such as e.g. HIV-1,HIV-2, HIV-3 (Journal of Infectious Diseases 194(2006)1677-1685,Molecular Medicine Today 1 (1995) 287-291, 1995), cytomegaloviruses(Journal of Virology 81(2007)9013-9023) or adenoviruses (Ophthalmologe105 (2008) 592-594, Ophthalmologe 97 (2000) 764-768) and for supportinghair growth (Archives of Dermatological Research 296 (6): 265-269, 2004,Annales de Dermatologie et de Venereologie 127(2000)769). Based on thewide range of therapeutically application, they are an importantsubstance class in medicine.

If cyclosporins and their derivatives are used for the treatment of saiddiseases, then they have in addition to the desired therapeutic effect,immunosuppressive effects, which is a drawback of many so far knowncyclosporin derivatives. In these cases, however, the immunosuppressiveeffect is an undesired side effect which should be eliminated. It isknown that cyclosporins and their derivatives are able to inhibitcyclophilins. But, if the cyclosporin compounds enter the cell, then thecomplexes of cyclosporin compounds and cyclophilin may inhibit theserine-threonine-phosphatase calcineurin. The inhibition of calcineurininduces then the undesired side effect of immunosuppression. But, thereexists also cyclophilins in the extracellular space whose inhibition bycyclosporin compounds induces desired therapeutic effects. Therefore, ifit is possible to provide cyclosporin compounds which show both aneffect in regard to the desired therapeutic purpose and are not able toenter cells, then the undesired side effects could be avoided.

Thus, it was the objective if the present invention to provide novelcyclosporin derivatives, which have the desired therapeutic effectswithout occurrence of the side effect of immunosuppression.Particularly; it was the objective of the present invention to provideactive agents, which are therapeutically effective, but cannot enterinto the cell.

Especially, it was one objective of the present invention to provideactive agents which are therapeutically effective against the followingdiseases without being immunosuppressive:

-   -   a) viral infection    -   b) acute and chronic inflammatory diseases    -   c) cancer    -   d) degenerative muscle diseases    -   e) neurodegenerative diseases, and    -   f) disorders, which are associated with an impairment of calcium        homeostasis,        wherein the viral infection can be caused by viruses such as        HIV, hepatitis A, hepatitis B, hepatitis C, hepatitis D, and        hepatitis E,        wherein the inflammatory disease includes preferably asthma,        chronic inflammations, chronic prostatitis, glomerulonephritis,        multiple chemical sensitivity, inflammatory intestinal diseases,        sepsis, inflammation of the vascular smooth muscle cells,        aneurysm, inflammation in the pelvic area, reperfusion injury,        rheumatoid arthritis, vasculitis, psoriasis, and ulcerative        colitis,        wherein the cancer disease preferably comprises lung cancer,        cancer of the bladder, hepatic cancer, pancreatic cancer, and        breast cancer,        wherein the degenerative muscle disease is preferably directed        to muscle dystrophy, collagen IV-myopathies, and the myocardial        reperfusion injury,        wherein the neurodegenerative disease is preferably selected        from Alzheimer's disease, Parkinson's disease, Huntington's        disease, multiple systemic atrophy, multiple sclerosis, cerebral        poliomyelitis, stroke, diabetic neuropathy, amyotrophic lateral        sclerosis, spinal cord injuries, and cerebral sclerosis,        wherein the disease associated with an impairment of calcium        homeostasis, refers preferably to myocardial infarct, stroke,        acute hepatic toxicity, cholestasis, and reperfusion injury of        transplanted organs, and

Surprisingly, it has been found that the derivatization of amino acid 1of cyclosporins with suitable imidazole, oxazole, or thiazolederivatives may change their features in a way that such changedcyclosporins have modified cell permeability. In particular introducingchemical groups in the abovementioned imidazole, oxazole, or thiazolederivatives according to the invention, such as e.g. an acid group, maychange the features of the cyclosporins in a way that they do notaccumulate in the extracellular space and do not enter into the cells.

An essential feature, which is obtained by derivatization ofcyclosporins with suitable imidazole, oxazole, or thiazole derivatives,is the ability of the novel cyclosporin-derivatives obtained in such away to inhibit furthermore the peptidyl-prolyl-cis/trans-isomeraseactivity of human cyclophilins, preferably with an IC₅₀ value of <100nMol without inhibiting thereby the serine-threonine-phosphatasecalcineurin and without entering into the cells.

The above mentioned objective is accomplished by the provision ofcompounds of the following formula I:

wherein A is an amino acid of the following formula H,

where the bonds that end at the wavy lines represent linkages to themoieties B and K and R₉ is a group of the following formula III, and

in which R₈ corresponds to a group of the following formula IV, or

in which R₈ corresponds to a group of the following formula V, or

in which R₈ corresponds to a group of the following formula VI, or

in which R₈ corresponds to a group of the following formula VII, or

in which R₈ corresponds to a group of the following formula VIII,wherein the residues L-R₂ and R₃ can be bound to the same or differentphenyl rings, or

in which R₈ corresponds to a group of the following formula IX, andwherein the residues L-R₂ and R₃ can be bound to the same or differentrings Q₂ or Q₃, or

in which R₈ corresponds to a group of the following formula X, whereinthe residues L-R₂ and R₃ can be bound to the same or different rings Q₂or Q₃, or

in which R₈ corresponds to a group of following formula XI, wherein theresidues L-R₂ and R₃ can be bound to the same or different rings Q₂ orQ₃, or

in which R₈ corresponds to a group of the following formula XII, whereinthe residues L-R₂ and R₃ can be bound to the same or different phenylrings, or

in which R₅ corresponds to a group of following formula XIII,

whereinthe bonds in the formulae IV to XIII that end at the wavy lines bind tothe CH₂— group of formula III on which R₈ is linked;whereinthe group of formula II is covalently bound by its CO to the alpha-aminoacid B in formula I via formation of an amide bond and the N—R₀ informula II is covalently bound to a carboxyl group of K via formation ofan amide bond, wherein

B is an amino acid selected from the following group:

-   -   alpha-aminobutanoic acid;    -   alanine;    -   threonine;    -   valine;    -   norvaline; and    -   alpha-aminobutanoic acid, alanine, threonine, valine, or        norvaline with a hydroxyl group modified side chain;

C represents sarcosine;

D is an amino acid selected from the following group:

-   -   leucine;    -   N-methylleucine;    -   valine;    -   gamma-hydroxy-N-methylleucine; and    -   gamma-hydroxyleucine;

E is an amino acid selected from the following group:

-   -   valine;    -   norvaline; and    -   a modified valine or norvalin having one of the carbon atoms of        the side chain substituted with a hydroxyl group;

F is an amino acid selected from the following group:

-   -   leucine,    -   N-methylleucine;    -   gamma-hydroxy-N-methylleucine; and    -   gamma-hydroxyleucine;

G is alpha-aminobutanoic acid or alanine;

H is D-alanine or D-serine;

I and J are amino acids, which are independently of each other selectedfrom the following group:

-   -   leucine;    -   N-methylleucine;    -   gamma-hydroxy-N-methylleucine; and    -   gamma-hydroxyleucine;

K is N-methylvaline or valine;

wherein

the amino acids B to K are linked to each other via formation of amidebonds;

wherein

the used symbols and indices have the following meanings:

X represents O, S, or N—R₁;

Y represents O, S, N—R₁, —CH₂— or —CH₂CH₂—;

A represents CH or N;

L represents a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—,—CH═CH—, —CH═CHCH₂—, —CH₂CH═CH—, —CH₂CH₂CH═CH—, —CH₂CH═CHCH₂—,—CH═CHCH₂CH₂, —OCH₂—, —OCH₂CH₂—, —OCH₂CH₂CH₂—, —OCH₂CH═CH—, —CONH—,—CONHCH₂— —CONHCH₂CH₂—, —CONHCH₂CH₂OCH₂CH₂—, —CONHCH₂CH₂OCH₂CH₂OCH₂CH₂—;

Q₁ represents together with the two atoms of the adjacent rings anaromatic or heteroaromatic ring from the group benzene, pyridine,pyrimidine, pyridazine, pyrazine, triazine, thiophene, furan, pyrrole,thiazole, isothiazole, oxazole, isoxazole, imidazole, pyrazole;

Q₂ represents together with the two atoms of the adjacent rings anaromatic or heteroaromatic ring from the group benzene, pyridine,pyrimidine, pyridazine, pyrazine, triazine, thiophene, furan, pyrrole,thiazole, isothiazole, oxazole, isoxazole, imidazole, pyrazole,thiadiazole, oxadiazole, triazole;

Q₃ designates each six membered ring in the middle of which it ispositioned;

R₀ represents H or CH₃;

R₁ represents H, (C1-C4)-alkyl or benzyl that can also be substitutedwith a —COOH group;

R₂ represents a polar deprotonizable group P_(s), or a group P_(s)′ thatunder physiological conditions can be converted to the group P_(s), or apolar protonizable group P_(b), or a group P_(b)′ that underphysiological conditions can be converted to the group P_(b);

R₃ represents H, (C1-C6)-alkyl, (C1-C6)-alkoxy, —OH, (C1-C6)-alkylthio,(C1-C6)-alkylsulfonyl, —SH, —CF₃, —COOH, —COO((C1-C6)alkyl), —CONH₂,—CONH((C1-C6)alkyl), —CON((C1-C6)alkyl)₂, nitro, halogen, cyano, amino,(C1-C6)alkyl-amino, (C1-C6)dialkyl-amino; R₃ can also be in the form ofa free electron pair, in particular, if R₃ binds to a heteroatom of thering Q₁, Q₂ or Q₃,

R₄ and R₅ are independently of each other H, (C1-C6)-alkyl,(C1-C6)-alkoxy, —CF₃, halogen or if R₄ and R₅ are situated on the ringin ortho position, then they can form together a —OCH₂O— or a —OCH₂CH₂O—group; R₄ and R₅ can also independently of each other be in the form ofa free electron pair; in particular, if R₄ or R₅ binds to a heteroatomof the ring Q₁,

R₇ represents H, —OH, —OCOOR₁₂, —OCOR₁₃, —OCONR₁₄R₁₅,—O-(tetrahydropyran-2-yl), —O-(tetrahydrofuran-2-yl), —O—CHR₁₆—OR₁₇,—SiR₁₈R₁₉R₂₀;

R₁₀ and R₁₁ represent independently of each other H, (C1-C6)-alkyl,—CF₃, halogen or can form together a —CH₂CH₂—, —CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂—, or —CH₂CH₂CH₂CH₂CH₂— group;

R₁₂ represents (C1-C6)-alkyl, benzyl or phenyl, wherein the alkyl groupcan be optionally substituted with fluorine or chlorine and the benzyland phenyl are optionally substituted with one or more substituent(s)from the group: (C1-C6)-alkyl, (C1-C6)-alkoxy, —CF₃, cyano, nitro orhalogen;

R₁₃ represents H or R₁₂;

R₁₄ and R₁₅ represent independently of each other H, (C1-C6)-alkyl,benzyl or phenyl, wherein the benzyl and phenyl can be optionallysubstituted with one or more substituent(s) from the group:(C1-C6)-alkyl, (C1-C6)-alkoxy, —CF₃, cyano, nitro or halogen;

R₁₆ represents H or (C1-C6)-alkyl;

R₁₇ represents (C1-C6)-alkyl, benzyl or phenyl; and

R₁₈, R₁₉ and R₂₀ represent independently of each other (C1-C6)-alkyl,benzyl or phenyl.

The invention includes also all pharmaceutically acceptable salts, aswell as tautomeric, enantiomeric and other stereoisomeric forms of thecompound of formula I.

According to this invention, if names of amino acids are used withoutthe nomenclature prefixes D- or L-according to Fischer, bothpossibilities are possible; but preferred is the isomer naturallypresent in the environment (mostly L-isomer). If a correspondingnomenclature prefix is used, then the corresponding isomer is preferredaccording to the invention.

The P_(s) and P_(b) groups are either ionic groups, i.e. anionic groupsP_(s) or cationic groups P_(b), or groups that in solutions easilydissociate to ionic groups P_(s) or P_(b). Preferably, these groupsexist partially or completely in ionic form in the pH range from 6 to 8.The P_(s) groups can also be polar groups having a hydrogen atom thatcould be cleaved off with stronger bases, wherein the hydrogen atom ispreferably bound to a heteroatom.

Examples of acid P₅ groups are the followings:

a) —COOH, —SO₃H, —CONH₂, —CONHNH₂, —SO₂NH₂, —PO₃H₂, —PO(OH)(NH₂),—CO(NHOH), —CO(NH(O—C1-C4-alkyl)), —CSNH₂, —NHCONH₂, —N(OH)CONH₂,—NHCO(NHOH), —NHCSNH₂, —CSNHNH₂;

b)

and R═—(C1-C4)-alkyl, —O(C1-C4)-alkyl, —NH(C1-C4)-alkyl,—NH(C1-C4-alkenyl), (substituted) aryl, (substituted) O-aryl,(substituted) NH-aryl, —CF₃ and other fluorinated (C1-C4-alkyl) groups;

c)

and R═—OH, —CN, —NO₂;

d)

and R═(C1-C4-alkyl), (substituted) aryl, —CF₃ and other fluorinated(C1-C4-alkyl) groups;

e)

and R═—(C1-C4-alkyl), —O(C1-C4-alkyl), —NH(C1-C4-alkyl),—NH(C1-C4-alkenyl), (substituted) aryl, (substituted) O-aryl,(substituted) NH-aryl, —CF₃ and other fluorinated (C1-C4-alkyl) groups;

f)

in which R═H, —(C1-C4-alkyl), —CF₃, and other fluorinated (C1-C4-alkyl)groups.

g) —OH and —SH, providing that L is a covalent single bond and that —OHor —SH can only be present as a substituent on a ring Q₁, Q₂ or Q₃ andthereby is bound to a carbon atom, which is positioned in the vicinityof a nitrogen atom.

According to the invention, it is especially preferred that the P_(s)group is one of the following groups, which preferably is directly boundvia a covalent bond to a heterocycle of formula IV-XII:

—COOH, —OH, —SH, —CONH₂, —CONHNH₂, —SO₃H, —SO₂NH₂, and the group

providing that, if P_(s) represents —OH or —SH, then L is a covalentsingle bond, and —OH or —SH is bound to a carbon atom of a ring Q₁, Q₂or Q₃, and said carbon atom is positioned in the vicinity of a nitrogenatom.

Examples of basic P_(b) groups are the following:

a)

wherein R_(a), R_(b) and R_(c) represent H and —(C1-C4-alkyl), R_(a) andR_(b) can be together —(CH₂CH₂)—, or —(CH₂CH₂CH₂)—, and R_(b) and R_(c)together with the nitrogen atom can be pyrrolidine, piperidine ormorpholine;

b)

wherein R_(a), R_(b), R_(c) and R_(d) represent H and —(C1-C4-alkyl),R_(a) and R_(b), R_(a) and R_(d), as well as R_(b) and R_(d) togethercan be —(CH₂CH₂)—, or —(CH₂CH₂CH₂)— and R_(b) and R_(c) together withthe nitrogen atom can be pyrrolidine, piperidine or morpholine;

c) an amino group selected from —NH₂, (C1-C6)alkyl-amino,(C1-C6)dialkyl-amino, pyrrolidine, pyperidine, morpholine or piperazine,which can be substituted at position 4 with (C1-C6)alkyl,—(C1-C6)alkanoyl, benzyl, benzoyl, or phenyl, wherein these substituentscan optionally have a —COOH group.

According to the present invention, it is especially preferred that theP_(b) group is one of the following groups, which is directly bound viaa covalent bond to a heterocycle of formula IV-XII:

The P_(s) and P_(b) groups can also be a

group,wherein R is an amino acid or an amino acid amide, which are bound viatheir amino groups, or a peptide consisting of 2-10 amino acids and inwhich the terminal amino acid can also be an amino acid amide. Preferredpeptide groups contain at least one amino acid from the group:D-glutamic acid, L-glutamic acid, D-aspartic acid, L-aspartic acid,D-glutamine, L-glutamine, D-asparagine, L-asparagine, D-cysteinesulfonic acid, L-cysteine sulfonic acid, D-arginine or L-arginine.Preferred are peptide groups of formula

In the present application, the term “amino acid” encompasses anyorganic compound containing an amino group and a carboxylic acid groupor a sulfonic acid group. In the present application, the naturallyoccurring amino acids are preferred.

The groups P_(s)′ and P_(b)′ are groups that can be converted underphysiological conditions to the groups P_(s) and P_(b), respectively.Compounds containing groups P_(s)′ and P_(b)′ are termed “prodrugs”.Such compounds do not necessarily have to be cell-impermeable, but theyare ideally transforming themselves to cell-impermeable derivativesafter application, preferably after oral application, so that theycannot penetrate into the cell at the site of action.

For example, carboxylic acid esters can be used as prodrugs forcarboxylic acids (P_(s)═—COOH) (K. Beaumont, et al., Current DrugMetabolism 4 (2003), 461-485). Examples of carboxylic acid estersP_(s)′═—COOR contain the residue R

a) (C1-C6-alkyl), (C2-C6-alkenyl), phenyl or benzyl, wherein thearomatic ring can have further substituents, preferably —CH₃, —CH₂CH₃,—CH₂CH₂CH₃, —CH(CH₃)₂, —CH₂CH₂CH₂CH₃, —CH(CH₃)CH₂CH₃, —CH₂CH═CH₂,benzyl;

b) (C1-C6-alkyl), in which the hydrogen atoms are replaced by ahalogene, preferably —CF₃, —CH₂CCl₃;

c) (C1-C6-alkyl), in which the hydrogen atom is replaced by —OH,(C1-C6-alkoxy) or a substituted amino group, preferably —CH₂OCH₃,—CH₂CH₂OCH₃, —CH₂CH₂OH, —CH₂CH₂N(CH₃)₂, —CH₂CH₂(morpholin-4-yl);

d)

wherein R_(e) represents H or (C1-C4-alkyl) and R_(f) represents(C1-C4-alkyl) or (C3-C6-cycloalkyl), preferably R_(e)═H, —CH₃, —CH₂CH₃,—CH(CH₃)₂ and R_(f)=—CH₃, —CH₂CH₃, —CH(CH₃)₂, —C(CH₃)₃, cyclohexyl;

e)

It is especially preferred that the Ps′ group is one of the followinggroups: —COOCH₃, —COOCH₂CH₃, —COOCH₂CH₂CH₃, —COOCH₂CH₂CH₂CH₃,—COOCH₂CH(CH₂CH₃)₂, —COOCH(CH₃)₂, —COOC(CH₃)₃, —COOCH₂CH₂N(CH₃)₂ or—COOCH₂CH₂(morpholin-4-yl).

Acetylated derivatives P_(b)′ can be used as prodrugs for basic P_(b)groups such as amidine and guanidine, wherein P_(b)′=

in which Z═O, S, NH or NCH₃ and R_(g)═(C1-C4-alkyl), (C3-C6-cycloalkyl)or benzyl, and preferably Z═O and R_(g)═—CH₃, —CH₂CH₃, —CH₂CH₂CH₃,—CH(CH₃)₂, benzyl.

It is especially preferred that R₂ is selected from the group ofresidues consisting of the following: —COOH, —OH, —SH, —CONH₂, —CONHNH₂,—SO₃H, —SO₂NH₂, —COOCH₃, —COOCH₂CH₃, —COOCH₂CH₂CH₃, —COOCH₂CH₂CH₂CH₃,—COOCH₂CH(CH₂CH₃)₂, —COOCH(CH₃)₂, —COOC(CH₃)₃, —COOCH₂CH₂N(CH₃)₂ or—COOCH₂CH₂(morpholin-4-yl), the groups

Further preferred is that R₀ represents —CH₃ in the inventive compoundsof general formula I.

Preferably, the group R₇ is selected from the group consisting of: —OH,—OCOCH₃, —OCOOCH₃, —OCH₂OCH₃, —Si(CH₃)₃ and —Si(CH₃)₂C(CH₃)₃. It isespecially preferred that R₇ represents —OH.

The bivalent group L is preferably selected from the group consisting ofa bond, preferably of a covalent single bond, —CH₂—, —CH₂CH₂—, —CH═CH—,—CONH— and —OCH₂—. It is especially preferred that L represents acovalent single bond.

It is further preferred that R₂ represents —COOH, —SO₃H, —SO₂NH₂,tetrazol-5-yl, —C═NH(NH₂) or —NH—C═NH(NH₂) and the bivalent group L is acovalent bond.

It is especially preferred that the group R₁ represents H or CH₃, andeven more preferred represents H.

Moreover, it is preferred that X represents N—R₁, even more preferred Xrepresents NH.

The ring Q₁ is particularly preferred benzene, pyridine or pyrimidine,and strongestly preferred benzene.

The rings Q₂ and Q₃ are independently of each other preferably benzene.If one compound contains both rings Q₂ and Q₃, then it is mostlypreferred that both rings Q₂ and Q₃ are benzene.

The bivalent moiety Y is especially preferred O, S, —CH₂—, or —CH₂CH₂—.

In addition it is preferred if R₃ is selected from the group whichconsists of H, —COOH, CH₃, OCH₃, F, Cl, Br and CN. Especially preferredR₃ represents H and F, and even more preferred H.

It is especially preferred that R₄ and R₅ are independently of eachother H or F, and even more preferred H.

It is especially preferred that group A represents CH.

It is especially preferred that groups R₁₀ and R₁₁ are independently ofeach other H or CH₃, an even more preferred H.

Moreover, it is preferred that the group R₈ is a group of formula IV, V,VII, IX or XI. It is especially preferred that group R₈ is a group offormula IV.

If group R₈ is a group of formula IV, then in a preferred embodiment Q₁represents benzene and X represents NH, L is a covalent single bond or—CH═CH—, R₂ is —COOH, —CONH₂, —CONHNH₂, —SO₃H, —SO₂NH₂, —COOCH₃,—COOCH₂CH₃, —COOCH₂CH₂CH₃, —COOCH₂CH₂CH₂CH₃, —COOCH₂CH(CH₂CH₃)₂,—COOCH(CH₃)₂, —COOC(CH₃)₃, —COOCH₂CH₂N(CH₃)₂ or—COOCH₂CH₂(morpholin-4-yl) or one of the groups

R₃ represents H, —COOH, —COOMe or F and preferably H; R₄ represents H orF, and preferably H, and R₅ represents H.

If group R₈ is a group of formula IV, then in a further preferredembodiment Q₁ represents pyridine or pyrimidine and X represents NH.Thereby, it is especially preferred that Q₁ is pyrimidine, L is acovalent single bond, R₂ represents SH, OH or NH₂, and preferably SH orOH, R₃ represents H, SH, OH or NH₂, and preferably H, SH or OH, and R₄and R₅ are preferably not present (since the heteroatoms of thepyrimidine ring are situated at this position).

If group R₈ is a group of formula IX, X or XI, then in a furtherpreferred embodiment X represents NH, A represents CH, Q₂ representsbenzene, L represents a covalent single bond, R₂ represents —SO₃H or—COOH and R₃ represents H.

If group R₈ is a group of formula XIII, then in a further preferredembodiment R₁, R₁₀ and R₁₁ represent H.

It is furthermore preferred that the scaffold of formula I is asubstituted cyclosporin A compound corresponding to the followingformula:

wherein R₉ has the same meanings as above or below defined.

Moreover, it is especially preferred that the compound of formula I is asubstituted cyclosporin A-derivative of following formula:

All the features of the compound of aforementioned formula I listed inthe different embodiments can be, according to the present invention,combined with each other provided that they are not mutually exclusive.If in the mentioned embodiments, residues of the compound ofaforementioned formula I are not explicitly defined in the mentionedembodiment, then they can have according to the present invention everyspecified general or particular definition.

As an example for compounds of aforementioned formula I the followingcompounds are listed:

Ac-CsA-aldehyde, TBDMS-CsA-aldehyde and Ac-CsA-acid,

which can serve as starting material for the synthesis of compounds offormula I derived from cyclosporin A:

Compounds 1, 2 and 3: Ac-CsA-aldehyde, TBDMS-CsA-aldehyde andAc-CsA-acid (CsA meaning cyclosporin A) (hereinafter):

wherein R₉ corresponds to the following structure:

and wherein in case of compound 1, PG represents an acetyl group (Ac)and W═H, in case of compound 2, PG represents a tert-butyldimethylsilylgroup (TBDMS) and W═H, and in case of compound 3, PG represents anacetyl group (Ac) and W═OH.

In the present invention, the following compounds 4 to 68 are especiallypreferred as compounds of formula I:

The groups listed above and below in this application are binding at theposition where the bond ends at a wavy line.

If tautomeric forms of compounds 4 to 68 are possible, said tautomericforms should also be covered by the indicated tautomer and they shouldbe object of the present application.

According to the current invention compounds 5, 13, 20, 22, 28, 31, 33,34, 35, 37, 42, 43, 45, 46 and 48, and in particular compounds 5 and 33have proved to be particularly preferred.

As used herein the term (C1-C4)-alkyl, respectively (C1-C6)-alkyl is tobe understood as a linear or branched alkyl group with 1 to 4,respectively 1 to 6 C-atoms, more preferably with 1 to 4 C-atoms, andeven more preferably with 1 to 3 C-atoms. Individual —CH— or —CH₂—groups can be replaced by N, NH, O or S. Likewise, one or more H-atomsof the alkyl group can be replaced by fluorine atoms. Examples of suchgroups are the following: methyl, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, s-butyl, t-butyl, 2-methyl-butyl, n-pentyl, s-pentyl,t-pentyl, n-hexyl, 2,2-(di-ethyl)-ethyl, trifluoromethyl,pentafluoroethyl and 2,2,2-trifluoroethyl, wherein methyl and ethyl arepreferred.

Within the present application, the term (C2-C6)-alkenyl is to beunderstood as a linear or branched group of hydrocarbon units with 2 to6 C-atoms, preferably with 2 to 4 C-atoms and especially preferred with2 to 3 C-atoms, wherein 1 or several double bonds, and preferably onedouble bond is present between two C-atoms. Individual —CH— or —CH₂—groups can be replaced by N, NH, O or S. Also, 1 or several H-atoms ofthe alkenyl group can be replaced by fluorine atoms. Examples of suchgroups are the following: ethenyl, propenyl, butenyl, pentenyl, hexenyl.The residues ethenyl and propenyl are especially preferred.

Within the present invention, the term —NH(C1-C4-alkenyl) is to beunderstood as an above defined (C1-C4)-alkenyl, which is bound via a—NH— unit. Same preferences as for (C1-C4)-alkenyl apply also here.

As used within the present invention, the term (C3-C6)-cycloalkyl is tobe understood as a cyclic hydrocarbon group with 3 to 6 cyclic C-atoms.Individual —CH₂— groups can be replaced by N, NH, O or S. Also, 1 orseveral H-atoms of the alkyl group can be replaced by fluorine atoms.Examples for such groups are the following: cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl. Cyclohexyl is especially preferred.

As used within the present invention, the term (C1-C6)-alkoxy,respectively —O(C1-C4-alkyl) is to be understood as an above defined(C1-C6)-alkyl respectively (C1-C4)-alkyl, which is bound via an oxygenatom. Herein, methoxy and ethoxy are especially preferred.

As used within the present invention, the term (C1-C6)-alkylthio is tobe understood as an above defined (C1-C6)-Alkyl, which is bound via asulfur atom. Herein, methylthio and ethylthio are particularlypreferred.

As used within the present invention, the term (C1-C6)-alkylsulfonyl isto be understood as an above defined (C1-C6)-alkyl, which is bound via asulfonyl group. Herein, methylsulfonyl and ethylsulfonyl are especiallypreferred.

According to the present invention, the term —COO((C1-C4)alkyl)) is tobe understood as an above defined (C1-C4)-alkyl, which is bound via a—COO-group, wherein the (C1-C4)-alkyl binds to the oxygen atom. Herein,—COO-methyl and —COO-ethyl are especially preferred.

According to the present invention, the term (C1-C6)alkyl-aminorespectively, —NH(C1-C4-alkyl) is to be understood as an above defined(C1-C6)-alkyl, respectively (C1-C4)-alkyl that binds via an —NH— unit.Examples include methylamino and ethylamino.

According to the present invention, the term (C1-C6)dialkyl-amino is tobe understood as a secondary amino group bearing the two (C1-C6)-alkyldefined as above. Examples include dimethylamino and diethylamino.

The term (substituted) aryl is understood within the present inventionas a mono- or polycyclic (preferably mono-, bi- or tricyclic) aromaticor heteroaromatic hydrocarbon residue with preferably 5, respectively 6to 10, and even more preferrably 5 to 6 aromatic ring atoms. Thearomatic or heteroaromatic hydrocarbon residue can carry furthermoresubstituents.

In this context the term aromatic respectively heteroaromatic unit isunderstood either as a single aromatic cycle, such as benzene,respectively a single heteroaromatic cycle such as pyridine, pyrimidine,thiophene, etc., or as a condensed aryl- or heteroaryl group, such asnaphthalene, quinoline, isoquinoline, benzothiophene, benzofuran andindole etc. Hence, according to the present invention examples of anaromatic or heteroaromatic unit are: benzene, naphthalene, furan,benzofuran, isobenzofuran, thiophene, benzothiophene, isobenzothiophene,pyrrole, indole, isoindole, pyridine, quinoline, isoquinoline, pyrazole,indazole, imidazole, benzimidazole, oxazole, 1,2-thiazole, 1,3-thiazole,benzothiazole, pyridazine, benzopyridazine, pyrimidine, benzopyrimidine,quinoxaline, pyrazine, phenazine, 1,2,3-triazole, 1,2,4-triazole,benzotriazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole,1,3,4-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole,1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,3,5-triazine, 1,2,4-triazine,1,2,3-triazine, tetrazole, 1,2,4,5-tetrazine, 1,2,3,4-tetrazine,1,2,3,5-tetrazine, purin, pteridine and indolizine. Preferred are hereinbenzene, naphthalene, furan, thiophene, pyrrole, pyrimidine, pyrazine,1,2,3-triazole, 1,2,4-triazole, benzotriazole, 1,2,3-oxadiazole,1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3-thiadiazole,1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,3,5-triazine,1,2,4-triazine, 1,2,3-triazine, tetrazole, 1,2,4,5-tetrazine,1,2,3,4-tetrazine, 1,2,3,5-tetrazine, purin, pteridine, and especiallypreferred benzene.

According to the present invention, the term (substituted) O-arylrespectively (substituted) NH-aryl is to be understood as an abovedefined (substituted) aryl that binds via an oxygen atom or a —NH— unit.

The substituent on the substituted aryl can be for example: methyl,ethyl, methoxy, ethoxy, fluoro, chloro, bromo, trifluoromethyl,pentafluoroethyl, —OH, —SH, —NH₂, —CN or —NO₂.

As above mentioned, a further embodiment of the present invention isdirected to a tracer-coupled compound, which comprises a compound ofaforementioned formula I and a tracer compound (tracer) that are linkedto each other via a covalent bond. The linkage of the compound of theformula I is present in a way that instead of a terminal atom or aterminal group of the compound of formula I a bond to the tracer,possibly through a linker, is present

The tracer is preferably covalently bound to the compound of formula I.However, the tracer can in principle be bound to the active agent in anymanner known to the skilled person as being suitable for the purpose ofthe invention. Especially preferred is that the tracer is covalentlybound via a linker to the compound of formula I.

Within the scope of this invention the linker can be any group that isknown to the skilled person as being suitable for the objective of theinvention. Preferably, it refers to a group, which is free of a proteasecleavage site. Especially preferred, the linker is selected from groupsforming an inter-atomic distance of four to 40 atoms, preferably 5 to 30atoms, and most preferably 6 to 20 atoms.

According to the present invention, under the term “tracer” areencompassed substances, such as dyes, voltage-sensitive indicators, pHindicators, calcium-sensitive indicators, radioactive elements,NMR-labels or electron spin labels, which were described several timesin the scientific literature (WO/2005/022158, EP 0649022, U.S. Pat. No.6,596,499, U.S. Pat. No. 7,090,995, U.S. Pat. No. 4,672,044). The termtracer comprises according to the present invention individual atoms ormolecules that are covalently bound to the active agent molecule.Thereby, one tracer and also several tracers can be directly, covalentlybound to the active agent molecule.

The tracer and also the several tracers can also be bound to amultifunctional linker or the tracer and also the several tracers can becovalently bound within an acidic peptide or terminally on an acidicpeptide.

Within the scope of this invention, “dyes” are substances that may beoptically verified by detection of the radiation emitted by them or bythe electromagnetic radiation not being absorbed by them. These includesfor example dyes like fluorescein isocyanate (FIC), fluoresceinisothiocyanate (FITC), (dimethylamino)naphthalene-S-sulfonyl chloride(DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissaminerhodamine B200-sulfonyl chloride (RB 200 SC) etc. A description ofnumerous suitable molecules can be found exemplarily in DeLuca,“Immunofluorescence Analysis”, in “Antibody As A Tool”, Marchalonis etal., Eds., John Wiley & Sons, Ltd., pp. 189-231, (1985).

Within the scope of this invention, “voltage-sensitive indicators” aresubstances, which depending on an applied electrical potentialdifference or on the present electrical potential change their physical,optical or catalytic properties thus, causing a detectable signal.Voltage-sensitive indicators such as DIBAC (Japanese Journal ofPharmacology 86(2001) 342-350, American Journal of Physiology—Heart &Circulatory Physiology 287(2004) H985-H993) are known to the skilledperson.

Within the scope of this invention, “pH indicators” are substances,which depending on the pH value change their physical, optical orcatalytic properties thus, causing a detectable signal. Such indicatordyes, like for example phenol red, bromothymol blue, bromophenol blueand many others are known to the skilled person.

Within the scope of this invention “calcium-sensitive indicators” aresubstances, which in the presence of calcium change their physical,optical or catalytic properties thus causing a detectable signal.Calcium-sensitive indicators known to the skilled person are for exampleaequorin and other calcium-sensitive dyes such as FURA-2.

“Radioactive elements” according to this invention generate for examplegamma radiation like for example the following isotopes ¹²⁴I, ¹²⁵I,¹²⁸I, ¹³¹I, ¹³²I or ⁵¹Cr, wherein ¹²⁵I is particularly preferred. Otherslike for example ¹¹C, ¹⁸F, ¹⁵O or ¹³N, can be detected by their positronemission and suitable detectors (positron-emission-radiography) andothers, like for example ¹¹¹In can be detected by electron capture.

According to this invention, “NMR-labels” are substances containingatoms with odd number of nucleons (sum of protons and neutrons). Suchatomic nuclei, for example ¹³C, ¹⁵N or ¹⁹F, posses a nuclear spin andtherefore, a nuclear magnetic moment.

“Electron spin labels” serve within the scope of this invention tomeasure the “electron paramagnetic resonance” using electron spinresonance. Hence, the microwave absorption of a sample is measured in anexternal magnetic field. Thus, molecules having a permanent magneticmoment (unpaired electrons) can be detected (Physics in Medicine &Biology. 43(1998)U 3-U 4, Clinical Chemistry & Laboratory Medicine.46(2008) 1203-1210).

The use of tracers is particularly advantageous if the tracer-coupledcompound of the aforementioned formula I is used in a diagnostic method(such as anamnesis inquiry, physical examination, use of imagingtechniques such as X-ray/MRI or analysis with laboratory values of theblood or other body fluids). If the compounds of formula I according tothe present invention further contain one or several tracers, thedistribution volume of the active agent can be detected based on saidtracers. Tracers can be furthermore used to quantify the active agent.

Such an—as an example mentioned—tracer-coupled compound according tothis invention is for example the following compound 69:

wherein R₉ corresponds to the following structure:

Another embodiment of the present invention is directed to a compound ofthe aforementioned formula I or the tracer-coupled compound according tothe present invention for use in medicine. In other words, a compound ofgeneral formula I is used as a pharmaceutically-active substancerespectively a drug, the tracer-coupled compound according to thepresent invention is used as a diagnostic agent in medicine. Theinventive compound can be used for the manufacture of any drug appearingto the skilled person as being suitable for the compound of the presentinvention. Preferably, the inventive compound is used for themanufacture of a non-immunosuppressive drug. Drugs are considered to benon-immunosuppressive, if they do not decrease the functions of theimmune system.

The drugs or the compounds according to the invention, respectively, (inthe following only called drugs) can thereby be administered in anyform, which is known to the skilled person as being suitable for theintended purpose. For example the drug can be used in a form selectedfrom the group consisting of injections, infusions, tablets, crèmes,gels, ointments, sprays, capsules, syrups, emulsions, powders, flour,suppositories, or similar. In this context, it is especially preferredthat the drug is used in form of sprays, ointments, injections ortablets. Thereby, additives are very often needed that convert the drugin a dosage form to be administrable.

Therefore, one further embodiment of the present invention is directedto a pharmaceutical preparation, which contains a compound of theaforementioned formula I and preferably one or several additives.

The choice of the additive depends hereby specifically on the type ofthe dosage form. Preferred are such additives, which are physiologicallyacceptable and per se not pharmaceutically active

The pharmaceutical preparation can be each pharmaceutical preparationknown to the skilled person as being suitable. In a preferred embodimentthe pharmaceutical preparation is a spray, ointment, injection ortablet.

Application range of the compounds of the aforementioned formula I, thepharmaceutical preparation according to the invention and thetracer-coupled compounds according to the invention can be therapy anddiagnostics of disease, but also cosmetics. Therapy is primarilyunderstood as meaning the therapy of disease of human and animals.

Special advantages of the compounds according to the aforementionedformula I, of the pharmaceutical preparations according to the inventionand of the tracer-coupled compounds according to the invention areinherent in the animal and human medicine, with the application ofsubstances on or in cell suspensions, tissue cultures, transplants orthe whole mammal.

The present invention is furthermore directed to the use of compounds ofthe aforementioned formula I respectively of the pharmaceuticalpreparation according to the invention, in particular of thetracer-coupled compounds according to the invention as agents fordiagnosis.

In a further embodiment, the present invention refers to a compoundaccording to the aforementioned formula I or a pharmaceuticalpreparation according to the invention for the treatment of thefollowing diseases:

-   -   a) viral infections    -   b) acute and chronic inflammatory diseases    -   c) cancer    -   d) degenerative muscle diseases    -   e) neurodegenerative diseases, and    -   f) disorders which are associated with an impairment of calcium        homeostasis.

In other words, the present invention is also directed to the use of acompound of the aforementioned formula I or the pharmaceuticalpreparation of the invention for the manufacture of a medicament for thetreatment of the diseases mentioned above under a) to f). In still otherwords, the present invention is directed to a method for the treatmentof one of the diseases mentioned above under a) to f) in an individualin need of treatment comprising the administration of a therapeuticallyeffective amount of a drug comprising/consisting of a compound accordingto the aforementioned formula I or the pharmaceutical preparation of theinvention. The individual to be treated is preferably a mammal. Mammalsmay be humans and animals.

In another embodiment, the present invention refers to a tracer-coupledcompound according to the invention for the diagnosis of the followingdiseases:

-   -   a) viral infections    -   b) acute and chronic inflammatory diseases    -   c) cancer    -   d) degenerative muscle diseases    -   e) neurodegenerative diseases, and    -   f) disorders which are associated with an impairment of calcium        homeostasis.

In other words, the present invention is also directed to the use of atracer-coupled compound of the invention for the manufacture of amedicament for diagnosis of the diseases mentioned above under a) to f).In still other words, the present invention is directed to a method forthe diagnosis of one of the diseases mentioned above under a) to f) inan individual in the need of treatment comprising the administration ofa diagnostically effective amount of a drug comprising/consisting of atracer-coupled compound according to the invention. The individual to betreated is preferably a mammal. Mammals may be humans and animals.

According to the invention, the aforementioned viral infection ispreferably caused by viruses such as HIV, hepatitis A, hepatitis B,hepatitis C, hepatitis D, and hepatitis E.

According to the invention, the aforementioned inflammatory diseaseincludes preferably asthma, chronic inflammations, chronic prostatitis,glomerulonephritis, multiple chemical sensitivity, inflammatoryintestinal diseases, sepsis, inflammation of the vascular smooth musclecells, aneurysm, inflammation in the pelvic area, reperfusion injury,rheumatoid arthritis, and vasculitis.

According to the invention the aforementioned cancer disease isunderstood as meaning preferably—but not exclusively—lung cancer, cancerof the bladder, hepatic cancer, pancreatic cancer, and breast cancer.

According to the invention, the aforementioned degenerative muscledisease is preferably directed to muscle dystrophy, collagenIV-myopathies, and the myocardial reperfusion injury.

According to the invention, the aforementioned neurodegenerative diseaseis preferably selected from: Alzheimer's disease, Parkinson's disease,Huntington's disease, multiple systemic atrophy, multiple sclerosis,cerebral poliomyelitis, stroke, diabetic neuropathy, amyotrophic lateralsclerosis, spinal cord injuries, and cerebral sclerosis.

According to the invention, the aforementioned disease associated withan impairment of calcium homeostasis, refers preferably to myocardialinfarct, stroke, acute hepatic toxicity, cholestasis, and reperfusioninjury of transplanted organs.

Compounds of the aforementioned formula I of the present invention arenot immunosuppressive, extracellular effective inhibitors of theenzymatic activity of extracellular cyclophilins. As such, the compoundsare suitable for the treatment and/or prevention of cyclophilin mediatedacute and chronic diseases. The compounds are preferably, but not only,suitable for the treatment or prevention of persistent or chronicinflammatory diseases, neurogenic inflammation as well as inflammationassociated fibrosis and edema formation. This includes, but is notlimited to acute inflammatory overreaction (burning, post-operativeinflammation), gastro intestinal inflammation (colitis, Addison'sDisease), sepsis, disease of the respiratory system (asthma, chronicobstructive pulmonary disease), inflammatory vasculopathies(atherosclerosis, reperfusion injury), rheumatoid arthritis,inflammatory skin diseases (psoriasis, atopic dermatitis), eye diseases(keratoconjunctivitis) and inflammatory diseases of the peripheral andcentral nervous system (Parkinson's disease, stroke, multiplesclerosis).

In another aspect, the present invention is directed to a method foraccumulation of active agents in an intracellular space of amulti-cellular object, comprising the steps:

-   -   Providing a compound according to the aforementioned formula I        or a tracer-coupled compound according to the invention;    -   Contacting one of said compounds with a multi-cellular object.

In particular, the accumulation within the method of the inventionrefers to an in-vitro-accumulation. This means that the multi-cellularobject is an object separated from the living organism.

The “extracellular space” should refer to all areas, which are outsideof the cytosol and the membrane enclosing the cytosol. For example, theculture medium present in cell suspension is included, too.

The multi-cellular object can be any object that consists of at leasttwo identical or different biological cells.

The term “biological cell” thereby comprises human, animal as well asplant and bacterial cells, as well as unicellular organisms. If thebiological cells are bacterial cells or unicellular organisms, then theterm “multi-cellular object” refers to a conglomeration of several cellsas, for example, a cell colony or a bacterial culture. If the biologicalcells are human or animal cells, then the term “multi-cellular object”refers to a separated body part, such as a transplant, in particular anorgan-, a cell-, an extremities- or a tissue-transplant, blood or ablood fraction, such as blood plasma or an in-vitro culture of humanand/or animal cells, as for example a two-dimensional tissue culture ora spheroid culture of cells. If the biological cells are plant cells,then the term “multi-cellular object” refers to a part of a plant, suchas for example leaves, root or stem but also to a whole plant.

According to the invention, the multi-cellular object is a separatedorgan or a body-part, blood or a blood fraction, a cell culture or aplant.

Furthermore, the present invention is directed to the use of a compoundof the below shown formula XIV, which derives from cyclosporin A andcovers compounds 1 to 3, for the preparation of a compound according tothe aforementioned formula I or of the tracer-coupled compound accordingto the present invention. In other words, the present invention is alsodirected to a method for the preparation of such a compound according tothe aforementioned formula I or tracer-coupled compounds according tothe present invention using a compound of formula XIV.

Compound of formula XIV:

wherein R₉ represents a residue of the following formula:

wherein R₈ represents —CHO or —COOH; andwherein PG represents an alcohol protective group. The protective groupPG is able to protect a hydroxyl group so that it remains unchangedduring the transformation of other functional groups in the molecule orthat the unprotected hydroxyl group does not disturb thesetransformations. Examples of said protective groups as well as methodsfor their introduction and their cleavage are described in P. G. M. Wutsand T. W. Greene: “Protective Groups in Organic Synthesis”, 4. edition,2006; chapter “Protection for the hydroxyl group”. Preferred protectivegroups are ether, in particular substituted ether such as e.g.methoxymethyl-, methylthiomethyl-, benzyloxymethyl-, tert-butoxymethyl-,2-methoxyethoxymethyl-, 2-(trimethylsilyl)ethoxymethyl-,2,2,2-trichloroethoxymethyl-, tetrahydropyranyl-, tetrahydrofuranyl-,1-ethoxyethyl-, 1-methyl-1-methoxyethyl-, 1-methyl-1-benzyloxyethyl-,2,2,2-trichloroethyl-, 2-(trimethylsilyl)ethyl-, allyl-, benzyl-,4-methoxybenzyl-, 3,4-dimethoxybenzyl-, diphenylmethyl- ortriphenylmethyl ether;silyl ether such as e.g. trimethylsilyl-, triethylsilyl-,triisopropylsilyl-, tert-butyldimethylsilyl-, tert-butyldiphenylsilyl-,triphenylsilyl- or diphenylmethylsilyl ether;ester such as ester of the formic, acetic, chloroacetic, dichloroacetic,trichloroacetic, trifluoroacetic, pivalic or benzoic acid;carbonate such as e.g. methyl-, 9-fluoroenylmethyl-, ethyl-,2,2,2-trichloroethyl-, 2-(trimethylsilyl)ethyl-, isobutyl-, allyl-,2-propenyl-, 4-nitrophenyl-, benzyl-, 4-methoxybenzyl- or3,4-dimethoxybenzyl carbonate.

Particularly preferred are ethers such as methoxymethyl ether ortetrahydropyranyl ether, silyl ether such as trimethylsilyl-(TMS),tert-butyldiphenylsilyl-(TBDPS) or tert-butyldimethylsilyl ether (TBDMS)or an ester protective group such as acetyl. Especially preferred areTBDMS and acetyl.

Derivatives of cyclosporin A of the general formula I, which havesubstituents R₈ of the formula IV to formula XIII can be preparedstarting from a compound of formula XIV and suitable aromatic,heteroaromatic, aliphatic or heteroaliphatic primary amines presentingan unsubstituted or substituted amino group (X═NR₁), a hydroxyl group(X=0) or a thiol group (X═S) at an adjacent carbon atom. Preferably, thealdehydes of the formula XIV (R₈═—CHO) can be used. In case of reactionof aldehydes of the formula XIV with the amines, said reaction iscarried out in an inert solvent such as e.g. ethanol, methanol,isopropyl alcohol, tetrahydrofuran (THF), dichloromethane, chloroform,dimethylformamide (DMF), N,N-dimethyl acetamide (DMA), acetonitrile, orethyl acetate, or also mixtures of said solvents, optionally also inpresence of water. Preferred solvents are methanol, acetonitrile or DMF.The reaction can take place at room temperature or at elevatedtemperature up to boiling of the reaction mixture. Optionally, anoxidizing agent is added for completing the reaction. Examples ofoxidizing agents are quinones such as2,3-dichloro-5,6-dicyano-14-benzoquinone (DDQ); diacetoxyiodobenzene;benzofuroxan; nitrobenzene; dimethylsulfoxide (DMSO); metallic compoundswith metals having a higher oxidation state such as e.g. bariummanganate, manganese dioxide, nickel oxide, lead tetraacetate,pyridinium chlorochromate, scandium(III) triflate, ytterbium(III)triflate, copper(II) triflate, manganese triacetate;

halogens such as e.g. iodine; tert-butyl hypochlorite;

sulphur compounds having a higher oxidation state such as oxone, sodiummetabisulfite, sodium hydrogen sulfite, potassium bisulfate, potassiummonopersulfate; N-bromosuccinimide; N-chlorosuccinimide;N-iodosuccinimide or oxygen of the air.

Preferred oxidizing agents are N-bromosuccinimide or atmospheric oxygen.The reaction of the amines with carboxylic acids of the formula XIV(R₅═—COOH) can be carried out in presence of strong acids such as e.g.polyphosphoric acid, in particular at elevated temperature. In onepreferred variant, the reaction is carried out in two steps, whereinfirstly, an amide is obtained starting from a carboxylic acid of formulaXIV and an amine with elimination of water, and using said amide, in asecond step the heterocyclic compound of formula I is prepared withrepeated elimination of water. For preparation of the amide, may be usedreagents that are known to a skilled person for forming an amide bondunder dehydration conditions, e.g. using(benzotriazol-1-yl)-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP) in presence of a base such as e.g. diisopropylethylamine(DIPEA). The second step can for example take place by heating with ananorganic acid chloride such as thionyl chloride or POCl₃, by heating inpresence of an acid such as p-toluene sulfonic acid in a solvent such astoluene or xylene, by heating in an organic carboxylic acid such asacetic acid or propionic acid or in presence of water eliminatingreagents such as e.g. dicyclohexylcarbodiimide (DCC). Compounds offormula I may also be transformed into other compounds of formula I, bytransforming substituents R₂, R₃, R₄, R₅, R₁₀ or R₁₁, which are bound togroups R₈ of the formula IV to formula XIII using standard reactions oforganic synthesis known to the skilled person. For example, nitro groupscan be reduced to amino groups. Amino groups can be converted tosulfonamides using sulfonyl chlorides or to amides using acyl chloridesor other activated carboxylic acid derivatives. Carboxylic acidsR₂═—COOH can be transformed to their esters using alkyl halogenides.Also, under similar reaction conditions, a compound of formula I, withX═NH, can be converted to a compound X═N(C1-C4-alkyl) or N-benzyl.Furthermore, carboxylic acids R₂=—COOH can be transformed tocorresponding carboxylic acid amides using primary or secondary aminesunder standard amide coupling conditions.

One embodiment (i) of the present invention is directed to apharmaceutical preparation containing a compound of formula I accordingto the invention or a tracer-coupled compound according to theinvention.

Another embodiment (ii) of the present invention is directed to a methodfor the treatment/diagnosis of the following diseases a) to f) in anindividual in need of a treatment comprising the application of atherapeutically effective dose of a drug comprising a compound accordingto formula I or an inventive pharmaceutical preparation according toembodiment (i):

-   -   a) viral infections    -   b) acute and chronic inflammatory diseases    -   c) cancer    -   d) degenerative muscle diseases    -   e) neurodegenerative diseases, and    -   f) disorders, which are associated with an impairment of calcium        homeostasis.

Another embodiment (iii) of the present invention is directed to amethod according to embodiment (ii) for the treatment of viralinfections caused by viruses such as HIV, hepatitis A, hepatitis B,hepatitis C, hepatitis D, and hepatitis E, asthma, chronicinflammations, chronic prostatitis, glomerulonephritis, multiplechemical sensitivity, inflammatory intestinal diseases, sepsis,inflammation of the vascular smooth muscle cells, aneurysm, inflammationin the pelvic area, peritonitis, reperfusion injury, rheumatoidarthritis, vasculitis, lung cancer, cancer of the bladder, hepaticcancer, pancreatic cancer, breast cancer, muscle dystrophy, collagenIV-myopathies, myocardial reperfusion injury, Alzheimer's disease,Parkinson's disease, Huntington's disease, multiple systemic atrophy,multiple sclerosis, cerebral poliomyelitis, stroke, diabetic neuropathy,amyotrophic lateral sclerosis, spinal cord injuries, cerebral sclerosis,myocardial infarct, stroke, acute hepatic toxicity, cholestasis,reperfusion injury of transplanted organs, asthma, psoriasis, atopicdermatitis and ulcerative colitis.

Another embodiment (iv) of the present invention is directed to a methodfor accumulation of active agents/diagnostic agents in an extracellularspace of a multi-cellular object, comprising the steps:

-   -   Providing an inventive compound according to formula I or a        tracer-coupled compound according to the invention;    -   Contacting one of said compounds with the multi-cellular object.

Another embodiment (v) of the present invention is directed to a methodfor the preparation of a derivative of cyclosporin A according to theinvention by reacting a compound of the following formula

wherein R₉ represents a residue of the following formula:

wherein PG represents an alcohol protective group and W is H or OH andthe respective residue is linked via the bond at which end a wavy lineis drawn in.

Another embodiment (vi) of the present invention is directed to a methodfor the preparation of a tracer-coupled compound by reaction of acompound according to formula I with a tracer containing compound.

FIGURES AND EXAMPLES

The present invention should now be described in more detail on thebasis of the following figures and examples. The figures and exampleshave only an illustrative character and should in no way limit the scopeof the present invention.

There are:

FIG. 1: FIG. 1 shows two diagrams, in which the correspondingconcentration of compounds 5 and 33 are plotted against the percent ofthe control. In other words the diagrams in FIG. 1 show the cellpermeability of compound 5 and 33, as well as cyclosporin A inJurkat-cells.

FIG. 2: Verification of the immunosuppressive effect of compound 5compared to cyclosporin A in proliferation assay.

FIG. 3: Influence of 200 μg of compound 5 on the number of theeosinophilic granulocytes (eosinophils) in bronchial lavage.

FIG. 4: Influence of 200 μg of compound 5 on the number of theeosinophilic granulocytes (eosinophils) in lung tissue.

FIG. 5: Influence of compound 5 on the number of the CD4-positiveT-cells in the bronchial lavage.

FIG. 6: Influence of compound 5 on the number of the CD4-positiveT-cells in the lung tissue.

EXAMPLES

The following abbreviations are used in the examples:

-   CsA cyclosporin A-   DBU 1,8-diazabicyclo[5.4.0]undec-7-ene-   DCM dichloromethane-   DIC N,N′-diisopropylcarbodiimide-   DIPEA diisopropylethylamine-   DMAP 4-(dimethylamino)pyridine-   DMEM Dulbecco's Modified Eagle Medium-   DMF N,N-dimethylformamide-   DMSO dimethylsulfoxide-   DTT dithiothreitol-   EDO ethylene dioxide-   ES electro spray-   FACS Fluorescence Activated Cell Sorter-   FCS Fetal Calve Serum-   FITC fluoresceinisothiocyanate-   Fmoc fluorenylmethoxycarbonyl-   FSC Forward Scatter-   HATU 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate-   HBSS Hank's Buffered Salt Solution-   HOAc acetic acid-   HPLC High-performance Liquid Chromatography-   MeOH methanol-   MS mass spectrum-   MTT (methylthiazol-2-yl)-diphenyltetrazolium bromide-   NMP N-methylpyrrolidone-   OVA ovalbumin-   Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-   PBS Phosphate Buffered Saline-   PMA phorbol myristate acetate-   PyBOP (benzotriazol-1-yl)oxytripyrrolidinophosphonium    hexafluorophosphate-   SSC Side Scatter-   TAM RA tetramethyl-6-carboxyrhodamine-   TBDMS tert-butyldimethylsilyl-   TFA trifluoro acetic acid-   THF tetrahydrofuran-   Trt trityl

Example 1 Synthesis of Compound 1 (acetyl-CsA-aldehyde)

A solution of sodium periodate (1.03 mg; 4.81 mmol) in water (7 ml) wascarefully added dropwise to a solution of acetyl-cyclosporin A (3.0 g;2.4 mmol) and ruthenium (III) chloride hydrate (25 mg; 0.125 mmol) in amixture of acetonitrile (30 ml) and water (4 ml). Subsequently, themixture was stirred overnight at room temperature. Then, ethyl acetate(225 ml) was added and extracted using saturated saline solution (3×111ml). The organic phase was dried over MgSO₄ and subsequently in vacuo.Compound 1 could be separated from the acetyl-CsA carboxylic acid(compound 3) formed as by product using flash-chromatography (300 gsilica gel, 0.043-0.063 mm; solvent: 0.1% acetic acid in ethyl acetate).A yield of 1.76 g (60%) of compound 1 was obtained.

Example 2 Synthesis of Compound 2 (TBDMS-CsA-aldehyde)

Step 1:

A solution of cyclosporin A (4.0 g; 3.33 mmol) in dry methylene chloride(20 ml) was cooled to −20° C. under a protective atmosphere of nitrogen.Subsequently, at this temperature 2,6-lutidine (1.43 g; 13.3 mmol) andtert-butyldimethylsilyl trifluoromethanesulfonate (1.76 g; 6.66 mmol)were slowly added one after the other. After stirring overnight at roomtemperature the same amount of the silyl compound was again added andfurther 3 hours stirred. The solvent was removed in vacuo, the remainderchromatographed (silica gel; dichloromethane/methanol 100:0-90:10) and4.2 g TBDMS-CsA were obtained.

Step 2:

Ruthenium(III)chloride hydrate (33 mg; 0.16 mmol) was added to asolution of TBDMS-CsA (4.2 g; 3.19 mmol) in a mixture of acetonitrile(80 ml) and water (10 ml). Subsequently, a solution of sodium periodate(1.36 g; 6.36 mmol) in water (30 ml) was slowly added dropwise. Afterstirring overnight it was filtered, the filtrate largely concentrated invacuum and mixed with ethyl acetate. The organic phase was washed withsaturated saline solution, dried over Na₂SO₄ and concentrated. Theremainder was chromatographed (gradient: cyclohexane/ethyl acetate) andthereby, TBDMS-CsA-aldehyde was obtained, which was used without furthercleaning in the following reactions. Yield: 78 g (64% over two steps);MS (ES) C₆₆H₁₂₁N₁₁O₁₃Si calculated 1304. found 1305 (M+H)⁺.

Example 3 Synthesis of Compound 3 (acetyl-CsA-carboxylic acid)

The compound was synthesized according to a literature procedure(Bioconjugate Chem 3 (1992), 32-36) by oxidation of acetyl-cyclosporin Awith sodium periodate/potassium permanganate.

Example 4 Synthesis of Compound 4

50 mg (0.041 mmol) compound 1 and 11.25 mg cis-diamino biotin in 20 mlMeOH were heated for 1 h under reflux and subsequently, stirred at roomtemperature overnight. After evaporation of the methanol, the remainderwas taken in 10 ml DCM, treated with 9 mg N-bromosuccinimide and then,stirred for 1 hour. The acetyl-protected product was subsequentlyseparated by preparative HPLC and lyophilized. Then, theacetyl-protecting group was removed with 3 ml 0.1M LiOH in 50%tetrahydrofuran and the final product was isolated by preparative HPLC.Yield: 10 mg (18%).

Example 5 Synthesis of Compound 5

Method A:

A solution of 100 mg (0.081 mmol) of compound 1 and 190 mg (1.215 mmol,15 eq) 3,4-diaminobenzoic acid in MeOH (10 ml) was stirred for 12-48hours until the completion of the reaction (control by analytical HPLC).Subsequently, it was filtered and the solid was washed with 10 ml MeOH.1.5 ml of the methanolic solution was separated for the next step andthe remaining of the filtrate was concentrated in vacuum. Theacetyl-compound was subsequently purified by preparative HPLC (column RPC18 250×25 mm; gradient water+0.05% TFA/acetonitrile+0.05% TFA) andobtained after lyophilization as a white solid. Yield: 70 mg (69%).

1.5 ml 0.2M LiOH were added to 1.5 ml of the methanolic solution of theaforementioned acetyl-compound (˜6.1 μmol) and mixed until the end ofthe hydrolysis (about 3 hours; control by analytical HPLC). Afteracidifying with diluted hydrochloric acid, compound 5 was purified bypreparative HPLC (column RP C18 250×25 mm; gradient water+0.05%TFA/acetonitrile+0.05% TFA), lyophilized and isolated as white solid.Yield: 7 mg (87%).

Method B:

Step 1:

An excess of 3,4-diaminobenzoic acid (4.86 g; 31.9 mmol) was added to asolution of TBDMS-CsA-aldehyde (2.78 g; 2.13 mmol) in methanol (10 ml)and the mixture was stirred overnight by directing a weak air flowthrough it. It was filtered, the filtrate was concentrated and theremainder was used without further purification in the next step. MS(ES) C₇₃H₁₂₅N₁₃O₁₄Si calculated 1436. found 1437 (M+H)⁺.

Step 2:

The remainder of the previous step was mixed with a 1M-solution oftetrabutylammonium fluoride in THF (15 ml; 15 mmol) and the mixture wasstirred for 2.5 hours until the TBDMS-compound could not be anymoredetected. The solution was used directly, without separation of thesolvent, for separation by RP-HPLC (column C18; gradient H₂O (0.1%TFA)/MeOH (0.1% TFA)), and compound 5 was obtained after lyophilizationas a colorless solid. Yield: 909 mg (32% over two steps); MS (ES)C₆₇H₁₁₁N₁₃O₁₄ calculated 1322. found 1323 (M+H)⁺.

Example 6 Synthesis of Compound 6

A mixture of acetyl-CsA-carboxylic acid (50 mg; 0.04 mmol), methyl3-amino-4-hydroxy benzoate (10 mg; 0.06 mmol),benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP) (23 mg; 0.044 mmol) and DIPEA (21 μl) in DMF (3 ml) was stirredovernight and subsequently, the intermediate carboxamide was purified bypreparative HPLC. The amide was dissolved in toluene (10 ml), treatedwith thionylchloride (3 μl) and the solution was heated under reflux forseveral hours. After a new addition of thionylchloride (10 μl), followedby further heating for 24 hours, HPLC analysis showed a conversion tobenzoxazole of approximately 80%. The solvent was removed in vacuo andthe remainder was treated with MeOH (1 ml) and 0.2M NaOH (1 ml). Themixture was cooled to 5° C. and stirred overnight. It was acidified with18% solution of chlorhydric acid (100 μl) and compound 6 was isolated bypreparative HPLC purification.

Example 7 Synthesis of Compound 7 and Compound 8

Method A:

A solution of compound 5 (10 mg; 0.00732 mmol) in dry THF was treatedwith 17 mg CH₃I and 5-6 mg benzyltriethylammonium chloride and shackedovernight at room temperature. After addition of water, the pH wasadjusted to approximately 2-3 and the solution was subsequentlylyophilized under vacuum. 0.1M LiOH (4 ml) and MeOH (4 ml) were added,and the mixture was stirred for two hours. In addition to compound 7, bypreparative HPLC was also compound 8 isolated. Yield: compound 7: 3.5 mg(36%) and compound 8: 1 mg (9%).

Method B:

Compound 7 was prepared as compound 5 (method B), with the differencethat the following substances in the following amounts were used:TBDMS-CsA-aldehyde (300 mg; 0.23 mmol) and methyl 3,4-diamino benzoate(38 mg; 0.23 mmol); Yield: 52.3 mg (17%), colorless solid; MS (ES)C₆₈H₁₁₃N₁₃O₁₄ calculated 1336. found 1337 (M+H)⁺.

Example 8 Synthesis of Compound 9

Method A:

Compound 9 was prepared as compound 5 (method B), with the differencethat the following substances in the following amounts were used:TBDMS-CsA-aldehyde (300 mg; 0.23 mmol) and ethyl 3,4-diamino benzoate(42 mg; 0.23 mmol); Yield: 46 mg (15%), colorless solid; MS (ES)C₆₉H₁₁₅N₁₃O₁₄ calculated 1350. found 1351 (M+H)⁺.

Method B:

25 mg of compound 5 were dissolved in dry THF, treated with 20 mg ethylbromide, 8 mg K₂CO₃ and 5-6 mg benzyltriethylammonium chloride andshacked for 12-48 hours till the completion of the reaction (control byanalytical HPLC). Then, the mixture was concentrated in vacuum andseparated by preparative HPLC. The acetyl group was cleaved with 4 ml0.1M NaOH in 50% THF at room temperature within 2 h and the finalproduct was isolated by preparative HPLC. Yield: 10 mg (40%).

Example 9 Synthesis of Compound 10

Compound 10 was prepared as compound 9 in example 8 (method B), with thedifference that 1-bromo-2-ethyl butane was use instead of ethyl bromideas alkylating reagent.

Example 10 Synthesis of Compound 11

7 mg of compound 11 were prepared starting from 25 mg of compound 5 andthe added 1-bromobutane according to the procedure indicated at example7 (method A). Yield: 7 mg (28%).

Example 11 Synthesis of Compound 12

5 mg of compound 12 were prepared starting from 25 mg of compound 5 andthe added iso-butyl bromide according to the procedure indicated atexample 7 (Method A). Yield: 5 mg (20%).

Example 12 Synthesis of Compound 13

The dipeptide H-D-Glu(Ot-Bu)-D-Glu(Ot-Bu)-OH was bound on a2-chloro-tritylchloride resin via standardfluorenylmethoxycarbonyl(Fmoc)-peptide-synthesis. 152 mg ofdipeptide-resin, followed by DIPEA (10 μl) were added to a solution of13 mg of compound 5 and 4 mg HATU in a mixture of DMF (1 ml) and DCM (1ml). The reaction mixture was stirred for one hour at room temperature,then the solid was filtrated and washed with DMF (3×), followed by DCM(3×). Compound 13 was cleaved from the resin by stirring for 2 h at 5°C. with 2 ml of 100% trifluoroacetic acid (TFA). After TFA removal invacuum, the final product was purified by preparative HPLC. Yield: 4 mg(27%).

Example 13 Synthesis of Compound 14

A solution of acetyl-CsA-aldehyde (compound 1; 20 mg) andtert-butyl-3-(3,4-diamino-phenyl)-acrylate (50 mg; 0.21 mmol) in MeOH (1ml) was shacked at room temperature till completion of the reaction(control by analytical HPLC). The reaction mixture was cooled at 5° C.,0.2M NaOH (1 ml) was added and the reaction mixture was shacked at 5° C.till completion of the hydrolysis (control by analytical HPLC). Then,the reaction mixture was acidified with 18% chlorhydric acid (100 μl)and the product was purified by preparative HPLC. Yield: 5 mg (22.2%).

Example 14 Synthesis of Compound 15

Trifluoroacetic acid (1 ml) was added to ˜2.5 mg of compound 14. Themixture was shacked at room temperature for 30 min. After evaporation ofTFA in vacuum, the product was purified by preparative HPLC. Yield: 0.3mg (11.1%).

Example 15 Synthesis of Compound 16

A solution of acetyl-CsA-aldehyde (compound 1; 10 mg; 8 μmol) and4,5-diamino-2,6-dimercaptopyrimidine (6.9 mg; 40 μmol) in MeOH (1 ml)was shacked at room temperature till completion of the reaction (controlby analytical HPLC). Acetate hydrolysis, work-up of the reaction mixtureand purification of the compound 16 were performed in the same manner asdescribed for compound 14. Yield: 3.1 mg (28.8%).

Example 16 Synthesis of Compound 17

A solution of acetyl-CsA-aldehyde (compound 1; 10 mg; 8 μmol) and4,5-diamino-6-hydroxy-2-mercaptopyrimidine (3.2 mg; 18 μmol) in DMF (1ml) was stirred at 100° C. overnight. After removal of the solvent invacuum, acetate hydrolysis, work-up of the reaction mixture and thepurification of compound 17 were performed in the same manner asdescribed for compound 14. Yield: 1.5 mg (14%).

Example 17 Synthesis of Compound 18

A solution of acetyl-CsA-aldehyde (compound 1; 10 mg; 8 μmol) and5,6-diamino-1H-pyrimidin-4-one hemisulfate (7.3 mg; 21 μmol) in MeOH (1ml) was shacked at room temperature till completion of the reaction(control by analytical HPLC). Acetate hydrolysis, work-up of thereaction mixture and purification of the compound 18 were performed inthe same manner as described for compound 14. Yield: 0.83 mg (8%).

Example 18 Synthesis of Compound 19

Compound 19 was prepared in the same manner as described in example 17with the difference that 3,4-diaminobenzoic acid hydrazide (4.4 mg; 26μmol) was used as diamine. Yield: 2.5 mg (23.4%).

Example 19 Synthesis of Compound 20

Compound 20 was prepared in the same manner as described in example 17with the difference that 2,3-diamino-4,5-difluoro-benzene sulfonic acid(3.4 mg; 15 μmol) was used as a diamine. Yield: 2.5 mg (22.4%).

Example 20 Synthesis of Compound 21

Compound 21 was prepared in the same manner as described in example 17with the difference that 2,3-diamino-5-fluoro benzoic acid (3.8 mg; 22μmol) was used as diamine. Yield: 7.6 mg (70.9%).

Example 21 Synthesis of Compound 22

Method A:

Compound 22 was prepared as compound 5 (method B), with the differencethat the following substances in the following amounts were used:TBDMS-CsA-aldehyde (300 mg; 0.23 mmol) and 3,4-diaminobenzene sulfonicacid (51.8 mg; 0.275 mmol); Yield: 116.1 mg (37%), colorless solid; MS(ES) C₆₆H₁₁₁N₁₃O₁₅5 calculated 1358. found 1359 (M+H)⁺.

Method B:

A solution of 10 mg (8 μmol) of compound 1 and 4.9 mg (26 μmol)3,4-diaminobenzene sulfonic acid in 1 ml MeOH was shacked at roomtemperature till completion of the reaction (control by analyticalHPLC). The reaction mixture was cooled to 5° C. and 0.2M NaOH (1 ml) wasadded for cleavage of the acetyl protecting group. The reaction mixturewas shacked at 5° C. till completion of the hydrolysis (control byanalytical HPLC). Then, 100 ml 18% HCl were added and the product waspurified by preparative HPLC. Yield: 2.3 mg (21.1%).

Example 22 Synthesis of Compound 23

Method A:

3,4-Diaminobenzamide was obtained following a literature procedure (J.Med. Chem. 48 (2005), 1873-1885) and the diamine (35 mg; 0.23 mmol) wasreacted with TBDMS-CsA-aldehyde (300 mg; 0.30 mmol) in the same manneras described for compound 5 (method B). Yield: 43.7 mg (14%), brownishsolid; MS (ES) C₆₇H₁₁₂N₁₄O₁₃ calculated 1321. found 1322 (M+H)⁺.

Method B:

Compound 23 was prepared in the same manner as described in example 17with the difference that 3,4-diaminobenzonitrile (2.3 mg; 17 μmol) wasused as diamine. Yield: 0.4 mg (3.8%).

Example 23 Synthesis of Compound 24

A solution of acetyl-CsA-aldehyde (compound 1; 10 mg; 8 μmol) and5,6-diaminouracil sulfate (3.2 mg; 14 μmol) in DMF (1 ml) was stirredovernight at a temperature of 100° C. After solvents removal in vacuum,acetate hydrolysis, work-up of the reaction mixture and purification ofcompound 24 were performed in the same manner as described for compound14. Yield: 1.7 mg (16.2%).

Example 24 Synthesis of Compound 25

Compound 25 was prepared as compound 5 (method B) with the differencethat the following substances in the following amounts were used:iso-propyl 3,4-diaminobenzoate was prepared following a literatureprocedure (US2007/0032525) and the ester (45 mg; 0.23 mmol) was reactedwith TBDMS-CsA-aldehyde (300 mg; 0.23 mmol); Yield: 99.1 mg (32%),colorless solid; MS (ES) C₇₀H₁₁₇N₁₃O₁₄ calculated 1364. found 1365(M+H)⁺.

Example 25 Synthesis of Compound 26

Step 1:

To 3,4-diaminobenzoic acid (300 mg; 1.97 mmol) concentrated sulfuricacid (2 ml) was added, followed by 2-(dimethylamino)ethanol (176 mg;1.97 mmol). The mixture was stirred for 1 hour at room temperature,subsequently placed on ice and brought to pH 14 with a 20% sodiumhydroxide solution. Then, it was extracted with ethyl acetate, theorganic phase was dried over MgSO₄, evaporated to dryness and theremainder containing 2-(dimethylamino)ethyl-3,4-diaminobenzoate (154 mg;35%) was used without further purification in the next step.

Step 2:

The compound 26 was prepared in the same manner as compound 5 (method B)starting from TBDMS-CsA-aldehyde (300 mg; 0.23 mmol) and2-(dimethylamino)ethyl-3,4-diaminobenzoate (52 mg; 0.23 mmol). Yield:4.3 mg (1.3%), colorless solid; MS (ES) C₇₁H₁₂₀N₁₄O₁₄ calculated 1393.found 1394 (M+H)⁺.

Example 26 Synthesis of Compound 27

Step 1:

2-(Morpholin-4-yl)ethyl 3,4-diaminobenzoate was prepared as described instep 1 of the example 25 starting from 3,4-diaminobenzoic acid (300 mg;1.97 mmol) and 2-(morpholin-4-yl)ethanol (258 mg; 1.97 mmol). Yield: 61mg (12%).

Step 2:

The compound 27 was prepared in the same manner as compound 5 (method B)starting from TBDMS-CsA-aldehyde (300 mg; 0.23 mmol) and2-(morpholin-4-yl)ethyl-3,4-diaminobenzoic acid (61 mg; 0.23 mmol).Yield: 8.5 mg (2.6%), colorless solid; MS (ES) C₇₃H₁₂₂N₁₄O₁₅ calculated1435. found 1436 (M+H)⁺.

Example 27 Synthesis of Compound 28

Compound 28 was prepared as compound 5 (method B), with the differencethat the following substances in the following amounts were used:TBDMS-CsA-aldehyde (300 mg; 0.23 mmol) and 2,3-diaminobenzoic acid (35mg; 0.23 mmol); Yield: 65 mg (21%), colorless solid; MS (ES)C₆₇H₁₁₁N₁₃O₁₄ calculated 1322. found 1323 (M+H)⁺.

Example 28 Synthesis of Compound 29

Compound 29 was prepared as compound 5 (method B), with the differencethat the following substances in the following amounts were used:TBDMS-CsA-aldehyde (300 mg; 0.23 mmol) and methyl 2,3-diaminobenzoate(38 mg; 0.23 mmol); Yield: 50.8 mg (17%), colorless solid; MS (ES)C₆₈H₁₁₃N₁₃O₁₄ calculated 1336. found 1337 (M+H)⁺.

Example 29 Synthesis of Compound 30

Dimethyl 3,4-diaminophthalate was prepared following a literatureprocedure (J. Heterocyclic Chem. 10 (1973), 891-898) and the ester (30mg; 0.13 mmol) was reacted with TBDMS-CsA-aldehyde (175 mg; 0.13 mmol)in the same manner as described for compound 5 (method B). Yield: 38.6mg (21%), colorless solid; MS (ES) C₇₀H₁₁₅N₁₃O₁₆ calculated 1394. found1395 (M+H)⁺.

Example 30 Synthesis of Compound 31

Step 1:

An ethanolic solution of Na-ethanolate, prepared from sodium (181 mg,7.87 mmol) in ethanol (5 ml), was added to a suspension of dimethyl3,4-diaminophthalate (820 mg; 3.66 mmol) in ethanol (3.5 ml) and water(0.15 ml) and the mixture was heated at reflux during two hours. Aftercooling, the solid was filtered, dissolved in water (7 ml) and thesolution was neutralized with 1M HCl. Then, it was lyophilized and theremainder was stirred with methanol (50 ml). After renewed filtration,the filtrate was concentrated in vacuum and the 4,5-diaminophthalic acidwas obtained from the remainder after RP-HPLC (column C18; gradientH₂O/MeOH 95:5) and lyophilization. Yield: 553 mg (77%).

Step 2:

The compound 31 was prepared in the same manner as compound 5 (method B)starting from TBDMS-CsA-aldehyde (300 mg; 0.23 mmol) and4,5-diaminophthalic acid (45 mg; 0.23 mmol). Yield: 16.5 mg (5.3%),colorless solid; MS (ES) C₆₈H₁₁₁N₁₃O₁₆ calculated 1366. found 1367(M+H)⁺.

Example 31 Synthesis of Compound 32

Dimethyl 3,4-diaminophthalate was prepared following a literatureprocedures (J. Heterocyclic Chem. 10 (1973), 891-898) and the ester (51mg; 0.23 mmol) was reacted with TBDMS-CsA-aldehyde (300 mg; 0.23 mmol)in the same manner as described for compound 5 (method B). Yield: 63.9mg (20%), colorless solid; MS (ES) C₇₀H₁₁₅N₁₃O₁₆ calculated 1394. found1395 (M+H)⁺.

Example 32 Synthesis of Compound 33

5-(3,4-Diaminophenyl)tetrazol dihydrochloride was prepared following aliterature procedure (EP1944311) and the tetrazole (57 mg; 0.23 mmol)was reacted with TBDMS-CsA-aldehyde (300 mg; 0.23 mmol) in the samemanner as described for compound 5 (method B). Yield: 32.9 mg (11%),colorless solid; MS (ES) C₆₇H₁₁₁N₁₇O₁₂ calculated 1346. found 1346 (M⁺).

Example 33 Synthesis of Compound 34

Compound 34 was prepared as compound 5 (method B), with the differencethat the following substances in the following amounts were used:TBDMS-CsA-aldehyde (174 mg; 0.13 mmol) and 3,4-diaminobenzenesulfonicamide (25 mg; 0.13 mmol); Yield: 14.5 mg (8%), colorless solid; MS (ES)C₆₆H₁₁₂N₁₄O₁₄S calculated 1357. found 1358 (M+H)⁺.

Example 34 Synthesis of Compound 35

3,4-diaminobenzamidine was prepared following a literature procedure(WO1999/24395) and the amidine (35 mg; 0.23 mmol) was reacted withTBDMS-CsA-aldehyde (300 mg; 0.23 mmol) in the same way as described forcompound 5 (method B). Yield: 86.9 mg (29%), colorless solid; MS (ES)C₆₇H₁₁₃N₁₅O₁₂ calculated 1320. found 1321 (M+H)⁺.

Example 35 Synthesis of Compound 36

3-Amino-4-(methylamino)benzoic acid was prepared following a literatureprocedure (WO2005/030704) and the diamine (39 mg; 0.23 mmol) was reactedwith TBDMS-CsA-aldehyde (300 mg; 0.23 mmol) in the same manner asdescribed for compound 5 (method B). Yield: 87.7 mg (29%), colorlesssolid; MS (ES) C₆₈H₁₁₃N₁₃O₁₄ calculated 1336. found 1337 (M+H)⁺.

Example 36 Synthesis of Compound 37

4-Amino-3-(methylamino)benzoic acid was prepared following a literatureprocedure (WO2000/020400) and the diamine (39 mg; 0.23 mmol) was reactedwith TBDMS-CsA-aldehyde (300 mg; 0.23 mmol) in the same manner asdescribed for compound 5 (method B). Yield: 80.2 mg (26%), colorlesssolid; MS (ES) C₆₈H₁₁₃N₁₃O₁₄ calculated 1336. found 1337 (M+H)⁺.

Example 37 Synthesis of Compound 38

N-(3,4-Diaminophenyl)trifluoroacetamide was prepared following aliterature procedure (WO2004/039318) and the diamine (51 mg; 0.23 mmol)was reacted with TBDMS-CsA-aldehyde (300 mg; 0.23 mmol) in the samemanner as described for compound 5 (method B). Yield: 23.7 mg (7.4%),colorless solid;

MS (ES) C₆₈H₁₁₁F₃N₁₄O₁₃ calculated 1389. found 1390 (M+H)⁺.

Example 38 Synthesis of Compound 39

Compound 39 was prepared in the same manner as described in example 17,with the difference that 3,3′-diaminobenzidin tetrahydrochloridedihydrate (18.3 mg; 46 μmol) was used as a diamine. Yield: 4.5 mg(40.6%).

Example 39 Synthesis of Compound 40

Compound 40 was prepared in the same manner as described in example 17,with the difference that 1,2,4,5-tetraaminobenzene tetrahydrochloride(5.4 mg; 19 μmol) was used as diamine. Yield: 4.3 mg (41.1%).

Example 40 Synthesis of Compound 41

Compound 41 was prepared in the same manner as described in example 17with the difference that 4-(4-methylpiperazin-1-yl)-1,2-diaminobenzene(4.1 mg; 20 μmol) was used as diamine. Yield: 2.3 mg (20.9%).

Example 41 Synthesis of Compound 42

A solution of acetyl-CsA-aldehyde (25 mg; 20 μmol) und ethylendiamine(1.5 μl) in dichloromethane (5 ml) was stirred for 1 h at roomtemperature, and then for 20 minutes at 5° C. N-bromosuccinimide (4 mg)was added subsequently and the solution was stirred overnight. Thesolvent was removed in vacuo and the remainder was taken in ethylacetate (30 ml). Then, it was subsequently washed with saturated NaHCO₃—(2×10 ml), 5% KHSO₄— (2×10 ml) and saturated sodium chloride-solution(2×10 ml). After drying over MgSO₄, the solvent was removed in vacuo andsubsequently, the remainder was taken in a solution of 0.1 M NaOH (4 ml)in the same volume of tetrahydrofuran. It was stirred for 2-5 hours tillcompletion of the reaction (control by analytical HPLC). The product wasseparated by preparative HPLC. Yield: 7 mg (28%).

Example 42 Synthesis of Compound 43

Acetyl-CsA-carboxylic acid (50 mg; 0.04 mmol), L-serine methylesterhydrochloride (7 mg) and (benzotriazol-1-yl)oxytripyrrolidinophosphoniumhexafluorphosphate (PyBOP) (23 mg; 0.044 mmol) were dissolved in DMF (3ml) and subsequently treated with DIPEA (21 μl). After stirring for 50min at room temperature, the intermediate product was separated bypreparative HPLC. Yield: 27 mg (50%).

27 mg of amide-intermediate product were dissolved in toluene (20 ml)and then triethylamine (20 μl) and methansulfonyl chloride (10 μl) wereadded. After removal of solvents in vacuo, the residue was treated with0.2 M NaOH (2 ml) and THF (2 ml) and the mixture was stirred tillcompletion of the reaction (control by analytical HPLC). The finalproduct was isolated by preparative HPLC. Yield: 7 mg (27%).

Example 43 Synthesis of Compound 44

Compound 44 was prepared in the same manner as described in example 17with the difference that (R,R)-(−)-trans-1,2-diaminocyclohexanehydrochloride (3.3 mg; 22 μmol) was used as diamine. Yield: 5.8 mg(56.4%).

Example 44 Synthesis of Compound 45

A mixture of acetyl-CsA-aldehyde (11 mg; 8.8 μmol) and5,6-diamino-naphthalen-1-sulfonic acid (2.8 mg; 12 μmol) in DMF (1 ml)was shacked at room temperature overnight. The solvent was removed invacuo and MeOH (1 ml) and 0.2 M NaOH (1 ml) were added to the remainder.The reaction mixture was shacked at 5° C. overnight and the product waspurified by preparative HPLC. Yield: 8.0 mg (71.0%).

Example 45 Synthesis of Compound 46

N-(3,4-diaminophenyl)guanidine hydrochloride was prepared following aliterature procedure (WO1998/045275) and the diamine (62 mg; 0.307 mmol)was reacted with TBDMS-CsA-aldehyde (400 mg; 0.307 mmol) in the samemanner as described for compound 5 (method B). Yield: 121.7 mg (30%),colorless solid; MS (ES) C₆₇H₁₁₄N₁₆O₁₂ calculated 1335. found 1336(M+H)⁺.

Example 46 Synthesis of Compound 47

Step 1:

A solution of 2,4-diaminonitrobenzene (8 g; 52.2 mmol) and DIPEA (9.9 g;76.6 mmol) in DCM (300 ml) was cooled to 0° C. and at this temperaturetrifluoromethanesulfonic anhydride (23 g; 81.5 mmol) was added dropwisewithin 90 min. Then, it was stirred overnight, diluted with DCM, washedwith saturated NaHCO₃ solution, dried over MgSO₄ and evaporated invacuo. The remainder was stirred with ether (3×400 ml). The combinedsupernatants were combined andN-(4-amino-3-nitrophenyl)trifluormethansulfonamide was obtained afterchromatography purification on silica gel by elution with DCM; Yield:12.2 g (82%).

Step 2:

To a solution of the above nitro compound (12.2 g; 42.8 mmol) in MeOH(150 ml) was added 10% Pd—C(1.2 g). The mixture was stirred at roomtemperature under H₂ atmosphere overnight, the solid was filtered andwas with MeOH. In vacuo concentration, chromatography on silica gel(gradient: DCM/MeOH) gaveN-(3,4-diaminophenyl)-trifluormethansulfonamide. Yield: 3.87 g (35%).

Step 3:

Compound 47 was obtained in the same manner as described for compound 5(method B) starting from N-(3,4-diaminophenyl)-trifluormethansulfonamide(40 mg; 0.157 mmol) and TBDMS-CsA-aldehyde (205 mg; 0.157 mmol). Yield:14 mg (6.3%), reddish solid; MS (ES) C₆₇H₁₁₁F₃N₁₄O₁₄S calculated 1425.found 1426 (M+H)⁺.

Example 47 Synthesis of Compound 48

Step 1:

A solution of 1,2-diamino-3-fluorobenzene (100 mg; 0.8 mmol) in 100%sulfuric acid (5 ml) was heated by microwave at 150° C. for 30 min andthe progress of the reaction was followed by HPLC. Then, the solutionwas stirred in 200 g ice and neutralized with 40% NaOH solution. Thelyophilized remainder was suspended in acetonitrile (100 ml). Afterfiltration and subsequent removal of the solvent, the3,4-diamino-5-fluoro-benzensulfonic acid was dissolved in water (5 ml)and purified over a Dowex Cl⁻. Yield: 148 mg (90%).

Step 2:

A solution of Ac-CsA-aldehyde (compound 1; 12 mg; ˜10 μmol) in DMF (3ml) was added to 3,4-diamino-5-fluoro-benzensulfonic acid (3.6 mg; 17.5μmol) in DMF (1 ml). After addition of a solution of potassiummonopersulfate (6.1 mg; ˜10 μmol) in water (0.1 ml), the mixture wasstirred. The reaction was completed after 3 hours and the remainder waspurified by preparative HPLC. Yield: 7 mg (49%).

MeOH (1 ml) and ice-cold 0.2M NaOH (1 ml) were added to the purifiedfraction and the mixture was shacked at 5° C. overnight. Then, it wasacidified and the remainder was purified over preparative HPLC. Yield:4.9 mg (72%).

Example 48 Synthesis of Compound 49 Step 1: Synthesis of hexapeptideH-(L-Pro)₆-OH

The hexapeptide H-(L-Pro)₆-OH was prepared on a 2-chloro-tritylchlorideresin by standard Fmoc solid-phase peptide synthesis protocol. In everysynthetic cycle, Fmoc protected L-proline was activated with PyBOP andDIPEA in DMF and coupled for 2 h. The Fmoc-protecting group was cleavedwith a 20% solution of piperidine in DMF by stirring the amine one timefor 5 min and another time for 15 min. After the last coupling, thepeptide was cleaved from the resin with 100% TFA and purified bypreparative HPLC.

Step 2:

DIPEA (5 μl; 29 μmol) was added to a solution of compound 5 (10 mg; 7.6μmol) and HATU (3.3 mg; 8.7 μmol) in NMP (1 ml). The mixture was shackedfor 5 min and subsequently treated with H-(L-Pro)₆-OH (10 mg; 16 μmol).After shaking the mixture for 2 h, the product was purified bypreparative HPLC. Yield: 8.7 mg (60%).

Example 49 Synthesis of Compound 50

Step 1: Synthesis of hexapeptide H-(L-Ala)₆-OH The hexapeptide wassynthesized starting from Fmoc-protected L-alanine analogously to thepreparation protocol of H-(L-Pro)₆-OH as described at step 1 of theexample 48.

Step 2:

Compound 50 was obtained starting from compound 5 (10 mg; 7.6 μmol) andH-(L-Ala)₆-OH (10 mg; 23 μmol) in the same way as described at example48 (step 2). Yield: 8.1 mg (61%).

Example 50 Synthesis of Compound 51

Step 1: Synthesis of hexapeptide H-(β-Ala)₆-OH The hexapeptide wassynthesized starting from Fmoc-protected β-alanine analogously to thepreparation protocol of H-(L-Pro)₆-OH as described at step 1 of theexample 48.

Step 2:

Compound 51 was obtained starting from compound 5 (10 mg; 7.6 μmol) andH-(β-Ala)₆-OH (10 mg; 23 μmol) in the same way as described at example48 (step 2). Yield: 4.2 mg (32%).

Example 51 Synthesis of Compound 52

DIPEA (5 μl; 29 μmol) was added to a solution of compound 5 (10 mg; 7.6μmol) and HATU (3.3 mg; 8.7 μmol) in NMP (1 ml) and the mixture wasshacked for 5 min. After addition of 2-(N,N-dimethylamino)ethylamine(2.3 μl; 21 μmol), the mixture was shacked for additional 2 h and theproduct was then purified by preparative HPLC. Yield: 7.9 mg (75%).

Example 52 Synthesis of Compound 53

Compound 53 was obtained starting from compound 5 (10 mg; 7.6 μmol) and4-(2-aminoethyl)morpholine (2.1 μl; 16 μmol) according to the methoddescribed in example 51. Yield: 9.0 mg (83%).

Example 53 Synthesis of Compound 54 Step 1:N-(2-(piperazin-1-yl)ethyl)-tritylamine

A solution of 1-(2-aminoethyl)-piperazine (100 μl; 0.76 mmol) andtriethylamine (106 μl; 0.76 mmol) in DCM (20 ml) was cooled at 5° C. andtreated with trityl chloride. The mixture was stirred at 20° C.overnight and after solvent removal, the product was purified bypreparative HPLC. Yield: 84 mg (30%).

Step 2: 4-[4-(2-Aminoethyl)piperazin-1-yl]-4-oxo-butanoic acid

To a solution of N-(2-(piperazin-1-yl)ethyl)-tritylamine (55 mg; 0.15mmol) in DMF (2 ml) were added succinic anhydride (30 mg; 0.30 mmol) andDIPEA (51 μl; 0.3 mmol). The mixture was stirred for 2 h and the product4-oxo-4-{4-[2-(trityl-amino)ethyl]piperazin-1-yl}-butanoic acid waspurified by preparative HPLC. After lyophilization, TFA (2 ml) was addedto this product and the mixture was stirred for 30 min. The TFA was invacuo distilled and the remainder was purified by preparative HPLC.Yield: 25 mg (36%).

Step 3

A solution of compound 5 (10 mg; 7.6 μmol), HATU (8 mg; 8.7 μmol) andDIPEA (10 μl; 29 μmol) in NMP (1 ml) was shacked for 5 min. Afteraddition of 4-[4-(2-aminoethyl)piperazin-1-yl]-4-oxo-butanoic acid (24mg; 106 μmol), the mixture was shacked for additional 2 h. The productwas purified by preparative HPLC. Yield: 18 mg (67%).

Example 54 Synthesis of Compound 55

3-Amino-2-(methylamino)benzoic acid was prepared following a literatureprocedure (WO2008/008431) and the acid (38 mg; 0.23 mmol) was reactedwith TBDMS-CsA-aldehyde (300 mg; 0.23 mmol) in the same manner asdescribed for compound 5 (method B). Yield: 60.3 mg (20%), colorlesssolid; MS (ES) C₆₈H₁₁₃N₁₃O₁₄ calculated 1336. found 1337 (M+H)⁺.

Example 55 Synthesis of Compound 56

Step 1:

3-(N-Acetyl-N-methyl-amino)-2-nitrobenzoic acid was prepared following aliterature procedure (WO1998/24771) and the acid (5.35 g; 22.46 mmol)was heated together 6N HCl (46 ml) at 120° C. in a bomb tube for 90 min.After distilling off the solvent, dilution with MeOH, absorption ondiatomaceous earth, and chromatography on silica gel (CHCl₃/MeOH19:1+0.5% HOAc), 3.4 (77%) 3-(methylamino)-2-nitrobenzoic acid wasobtained.

Step 2:

A solution of the above described nitro compound (3.35 g; 17.1 mmol) andhydrazine hydrate (2.5 ml; 51.2 mmol) in dry MeOH (100 ml) was carefullymixed portionwise with a suspension of Ni-Raney (3.4 g) in the samesolvent. The mixture was stirred for 30 min at 50° C., concentrated, andthe remainder was filtered over silica with MeOH to give 1.24 g (44%)2-amino-3-(methylamino)benzoic acid as a dark solid.

Step 3:

The benzoic acid of the above step (32 mg; 0.19 mmol) was reacted withTBDMS-CsA-aldehyde (250 mg; 0.19 mmol) in the same manner as describedfor compound 5 (method B). Yield: 89 mg (35%), brown solid; MS (ES)C₆₈H₁₁₃N₁₃O₁₄ calculated 1336. found 1337 (M+H)⁺.

Example 56 Synthesis of Compound 57

The dipeptide H-L-Glu(OCH₂Ph)-L-Glu(OCH₂Ph)-NH₂ was bound to the Rinkamide resin using standardfluorenylmethoxycarbonyl(Fmoc)-peptide-synthesis (activation ofFmoc-L-Glu(OCH₂Ph)-OH with PyBOP/DIPEA and cleavage of the Fmoc-groupwith 2% DBU in DMF and 2% piperidine in DMF). A solution of compound 5(16 mg; 0.012 mmol), HATU (7 mg; 0.18 mmol) and DIPEA (6 μl) in DMF (2ml) was added to the resin. After overnight shacking, the product of thereaction was cleaved from the resin by treatment during 1 h at roomtemperature with TFA/DCM 1:1 (2 ml). After filtration and dilution withtoluene (2×2 ml) the solvent was removed under vacuum. The final productwas separated from the remainder by preparative HPLC. Yield: 6 mg (28%).

Example 57 Synthesis of Compound 58

Compound 58 was obtained following the method described in example 56starting from Fmoc-L-Leu-OH, Fmoc-L-Arg(Pbf)-OH, compound 5 (35 mg;0.0265 mmol), HATU (11 mg; 0.029 mmol) and DIPEA (9 μl). After washingthe resin with DMF and DCM, compound 58 was cleaved with TFA andisolated from the remainder by preparative HPLC. Yield: 2 mg (5%).

Example 58 Synthesis of Compound 59

Compound 59 was obtained in the same manner as described at example 57starting from Fmoc-L-Pro-OH, Fmoc-L-Asn(Trt)-OH and compound 5 (35 mg;0.0265 mmol). Yield: 28 mg (69%).

Example 59 Synthesis of Compound 60

Compound 60 was obtained in the same manner as described at example 57starting from Fmoc-L-Cys(Trt)-OH, Fmoc-L-Ala-OH and compound 5 (35 mg;0.0265 mmol). The cleavage of the final compound from the resin wascarried out with DTT (100 mg) containing TFA (3 ml). Yield: 12 mg (30%).

Example 60 Synthesis of Compound 61

Compound 61 was obtained in the same manner as described at example 59starting from Fmoc-L-Cys(Trt)-OH and compound 5 (35 mg; 0.0265 mmol).Yield: 4.4 mg (11%).

Example 61 Synthesis of Compound 62

Compound 62 was obtained in the same manner as described at example 57starting from Fmoc-L-Ser(t-Bu)-OH, Fmoc-L-Glu(Ot-Bu)-OH and compound 5(35 mg; 0.0265 mmol). Yield: 11.6 mg (28%).

Example 62 Synthesis of Compound 63

Compound 63 was obtained in the same manner as described at example 57starting from Fmoc-L-Glu(Ot-Bu)-OH and compound 5 (35 mg; 0.0265 mmol).Yield: 13 mg (31%).

Example 63 Synthesis of Compound 64

To a solution of compound 5 (10 mg; 7.6 μmol) in NMP (1 ml) were addedHATU (3.3 mg; 8.7 μmol) and DIPEA (5 μl). After stirring of the mixturefor 2 min, 1-(2-aminoethyl)-piperazine (1.2 mg; 9.4 μmol) was added andthe mixture was shacked for 3 hours. Compound 64 was separated bypreparative HPLC. Yield: 3.7 mg (34%).

Example 64 Synthesis of Compound 65

DIC (2 μl) and DMAP (1.4 mg; 11.4 μmol) were added to a solution ofcompound 5 (10 mg; 7.6 μmol) in DCM (1 ml). After stirring for 30minutes, the solvent was removed in vacuo. The remainder was treatedwith a solution of salicylic acid (2 mg; 14.5 μmol) in NMP (1 ml), themixture was shacked overnight and then compound 65 was isolated bypreparative HPLC. Yield: 1.5 mg (13%).

Example 65 Synthesis of Compound 66

Step 1:

N-(Trityl)-2-(2-(2-aminoethoxy)ethoxy)ethylamine was prepared followinga literature procedure (Eur. J. Org. Chem. 2009, 3953-3963).

Step 2:

Compound 5 (50 mg; 0.038 mmol) and PyBOP (19.7 mg; 0.038 mmol) weredissolved in NMP (3 ml); DIPEA (19.3 μl) and the amine of step 1 (17.7mg; 0.045 mmol) were added. The reaction mixture was stirred for 1 hour,diluted with ethyl acetate (50 ml) and subsequently washed for two timeswith 5% KHSO₄—, 5% NaHCO₃— and saturated sodium chloride-solution. Theorganic layer was dried over MgSO₄, concentrated in vacuo and reactedwith TFA (2 ml). After 15 minutes at room temperature, the reactionmixture was concentrated and compound 66 was isolated by preparativeHPLC. Yield: 29 mg (52%).

Example 66 Synthesis of Compound 67

DIPEA (10 μl) was added to a solution of cis,cis-1,3,5-cyclohexanetricarboxylic acid (22 mg; 0.1 mmol), compound 66 (7.3 mg; 0.005 mmol)and PyBOP (3 mg; 0.0058 mmol) in NMP (1 ml). The mixture was stirred for2 hours at room temperature and compound 67 was isolated by preparativeHPLC. Yield: 5 mg (61%).

Example 67 Synthesis of Compound 68

DIPEA (10 μl) was added to a solution of succinic anhydride (20 mg; 0.2mmol) and compound 66 (7.3 mg; 0.005 mmol) in NMP (1 ml) and the mixturewas stirred overnight at room temperature. Compound 68 was then isolatedby preparative HPLC. Yield: 2.9 mg (37%).

Example 68 Preparation of Compound 69 by Derivatization of Compound 5with a Molecule of Dye

a) Synthesis of TAMRA-EDO (tracer):

A solution of 5(6)-carboxytetramethylrhodamine (20 mg; 46.5 μmol) in DMF(5 ml) was treated with2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (19 mg; 8.3 μmol) and stirred for 5 minutesfor pre-activation. To this solution 2,2-(ethylenedioxy)ethyldiamine (68μl; 465 μmol) was then added. After 2 h of stirring, the product wasisolated by preparative HPLC. Yield: 15 mg (57.6%).

b) Synthesis of compound 69:

Compound 5 (10 mg; 7.56 μmol) was dissolved in DMF (1 ml) and afteraddition of HATU (3.2 mg; 8.3 μmol) and DIPEA (3.9 μl; 22.68 μmol) wasstirred for 5 minutes for pre-activation. Then, the solution ofTAMRA-EDO (8.5 mg; 15.12 μmol) dissolved in DMF (1 ml) was added. After2 h of stirring, the product (compound 69) was isolated by preparativeHPLC. Yield: 7 mg (50%).

Example 69 Inhibition of the peptidyl-prolyl-cis/trans-isomerase(PPlase) activity of the cyclophilin Cyp18wt

The determination was performed on a UV/Vis spectrometer at 10° C.cuvette temperature, whereby the device is operated at 390 nm inkinetic-mode (measurement buffer: 35 mM HEPES/NaOH pH 7.8 (AppliChemA1069)). 0.58 nM recombinant human wild type Cyp18 (Cyp18 wt), 2.33 nMbovine serum albumin (BSA) and 1 mg/ml chymotrypsin (Merck) solved in 35mM HEPES/NaOH pH 7.8 as well as different concentration of each testsubstance, where appropriate, were in the cuvette. The reaction wasstarted with a stock solution of Suc-Ala-Ala-Pro-Phe-4-nitroanilide(Bachem, 10 mg/ml DMSO) as substrate so that a final substrateconcentration of 32 μM resulted. The extinction-time-curves wereevaluated after expiry of the fast phase of the reaction i.e. afterapproximately 60 seconds according to a rate equation of a first-orderreaction. IC₅₀ values of the inhibitors result from the first order rateconstant of the non-inhibited reaction in ratio to the inhibitedreaction (% inhibition) measured in function of the inhibitorconcentration in the cuvette. The resulting IC₅₀ values of the testcompounds are listed in table 1.

Example 70 Inhibition of the Phosphatase Activity of Calcineurin

The determination of the dephosporylating calcineurin activity inpresence and absence of inhibitors was carried out in ascintillation-proximity-assay (see also R. Baumgrass, et al.; J. Biol.Chem. 276 (2001), 47914-47921). The measurement was carried out byincubation of 10 μMol of the biotinylated phosphopeptideAsp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-pSer-Val-Ala-Ala-Glu(MW=2192.0) (RH-peptide) marked with ³³P in a sample buffer (40 mMTris-HCl, pH 7.5, 100 mM NaCl, 6 mM MgCl₂, 0.5 mM dithiothreitol, 1 mM.CaCl₂, 0.1 mg/ml bovine serum albumin) in presence of 50 nM calmodulinand 1.32 nM calcineurin at 30° C. for 20 min in a 96 well micro titerplate (Costar, Bodenheim, Germany). The total volume of the assay/wellwas 100 μl. After this reaction time a 90 μl aliquot of the reactionmixture was transferred into a scintillation cavity coated withstreptavidin and incubated for 20 min at 22° C. After a washing step theRII phosphopeptide-associated ³³P radioactivity was measured in aMicroBeta Top Counter (Wallac) and the enzyme activity was indicated asaverage value of three measurements±S.D. The uninhibiteddephosphorylation activity has been set on 100%. Thecalcineurin-inhibition of potential cyclophilin-inhibitor-complexes hasbeen measured in presence of 10 nM to 50 μM Cyp18 concentrations withconstant inhibitor concentration of 10-50 μM (typical stock solution 1mM in DMSO). The results for the test compounds are listed in Table 1.

Example 71 Verification of the Immunosuppressive Effect in the CellularNFAT-Reporter-Gene Assay

An antigen specific stimulation of the T-cell receptor causes theactivation of transcription factors such as e.g. NFAT (nuclear factor ofactivated T cells). NFAT-activation is essential for the expression ofinterleukine-2 and therefore, for the proliferation of T-cells as partof the immune response. A stable NFAT-reporter-gene cell line enablesthe analysis of modulators of the NFAT-pathways and thus, the evaluationof the immunosuppressive effects of compounds.

The stably transfected reporter-gene cell line GloResponse NFAT-RE-luc2PHEK293 (promega Corp. USA) was cultivated in DMEM (10% FCS; 2 mMGlutamin; 200 μg/ml Hygromycin) and seeded on 96-well plates (200.000cells/well). The compounds to be tested were either added to the cellsin a 2-point-determination (5 μM and 10 μM) or an IC_(H)-determination(concentration series 4.1 nM-1 μM). Unmodified cyclosporin A has beenused as immunosuppressive reference compound. Immediately thereafter thecells were stimulated with 5 nM PMA and 2 μM ionomycin and incubatedover 16 h (37° C.; cell incubator). This double stimulation simulates aT-cell receptor stimulation and causes a NFAT activation withsubsequently expression of the luciferase-reporter-gene. The amount ofluciferase was quantitatively determined after 16 h by measurement ofthe bioluminescence by Bright-Glo Luciferase-Assay-System (PromegaCorp., USA). The averaged luciferase values of the vehicle treated (1%DMSO) and with PMA/ionomycin stimulated cells were used as positivecontrol and reference value. The results for the test compounds arelisted in Table 1.

In the following table 1 the compounds 4-68 according to the inventionin comparison to cyclosporin A are listed in regard to their activityfor inhibition of Cyp18 and calcineurin as well as their activity in theNFAT-reporter-gene assay:

TABLE 1 Inhibition of Indirect Inhibition NFAT-reporter- Cyp18wt ofCalcineurin gene assay Compound IC₅₀ [nM] IC₅₀ [nM] IC₅₀ [nM]Cyclosporin A ~8 85 31.4 4 264 >10000 5 9.6 >10000 >10000 6 700 7 11.96900 >10000 8 90.4 9 13.9 7000 >10000 10 11.5 6900 >10000 11 13.0 470010000 12 31.4 2000 13 11.1 >10000 >10000 14 36.4 3200 154.8 >10000 >10000 16 15.1 7500 17 >50 >10000 18 1.4 10000 >10000 19 5.47000 >10000 20 6.7 >10000 >10000 21 22.2 >10000 5300 228.7 >10000 >10000 23 3.8 >10000 >10000 24 6.2 >10000 >10000 25 22.0 790010000 26 10.8 6200 >10000 27 5.9 6400 7400 28 60.0 >10000 >10000 2929.5 >10000 10000 30 29.1 10000 >10000 31 39.9 10000 >10000 3224.9 >10000 >10000 33 9.8 >10000 >10000 34 16.4 6400 >10000 35 12.04800 >10000 36 1000 >10000 >10000 37 61.5 >10000 >10000 38 14.3 33403800 39 6.1 >10000 10000 40 5.5 8000 >10000 41 3.0 >10000 >10000 42 57.79500 10000 43 45.3 10000 5300 44 9.2 >10000 >10000 45 3.2 >10000 >1000046 13.1 6630 >10000 47 13.2 >10000 >10000 48 8.7 >10000 49 30.9 >1000050 7.3 10000 51 7.3 >10000 52 8.0 >10000 53 12.7 4200 54 7.1 7200 55209.3 >10000 56 >1000 >10000 57 16.9 60 ~10 61 ~10 62 4.4 63 5.1 64 17.210000 65 20.2 >10000 66 15.0 >10000 67 8.4 >10000 68 8.5 >10000

Example 72 Determination of Cell Permeability in Jurkat Cells

Jurkat cells of the cell culture have been harvested and centrifuged.The medium has been discarded and the cells were washed once with RPM′(without phenol red, 10% FCS, Hepes, gentamicin). The cells werere-suspended in a medium adjusted to a cell concentration of1.0×10⁵-3.0×10⁵ cells/ml and subsequently incubated with a fluorescentCsA derivative (0.5 μM-2.0 μM) at 37° C., 5-10% CO₂ and 100% humidity.The cells were then centrifuged, the medium discarded and the cellpellet was washed once with medium and the cells were resuspended in anew medium. Subsequently, the cells were incubated for a half hour to 4hours with the corresponding concentration (0.01 μM to 100 μM) of thetest compound to be analyzed and then centrifuged again. The medium wasdiscarded; it was once washed with 1 ml PBS and suspended in 500 μl PBS.The analysis was carried out using FACS-measurement by quantification ofthe replaced fluorescent CsA-derivative. The reference (withoutinhibitor) was thereby set 100%. The results for compound 5 and forcyclosporin A of this test system show (FIG. 1), that compound 5 is notcell permeable in comparison to CsA.

Example 73 Determination of Cell Permeability in Caco2 Cell Membranes

The test substances were diluted from a 10 mM DMSO solution to a finalconcentration of 5 μM in HBSS buffer pH 7.4. Subsequently, incubationtakes place for 2 hours at 37° C. and 5% CO₂ on a differentiated monolayer of Caco2-cells, which were grown for 10 days on aTranswell-membrane. The concentration of the test substance wasdetermined in the starting—as well as in the receiving-well. Theapparent permeability (P_(app)) in apical to basolateral direction (A-B)and vice versa (B-A) was calculated according to the following formula:P_(app)=1/AC₀ (dQ/dt), wherein A is the area of the Transwell membrane,C₀ the substance concentration at the time point t=0 and dQ/dtrepresents the amount of substance migrated per time period. The resultsfor the test compounds are listed in table 2.

Example 74 Determination of Cell Permeability in PAMPA Cell Membranes(Parallel Artificial Membrane Permeability Assay)

The test substances were diluted from a 10 mM DMSO solution to a finalconcentration of 500 μM in Hepes buffer pH 7.4 and transferred to aTranswell membrane, which was covered with a membrane forming solutionof 10% 1,2-dioleyl-sn-glycer-3-phosphocholin and 0.5% (w/v) cholesterolin dodecane. After an incubation of 16 hours at room temperature in aliquid chamber the optical density of the solution after filtration hasbeen measured in a range of 250 to 500 nm in steps of 10 nm each. Thepercentage “Flux” has been calculated from the integral of the graphbetween 250 and 500 nm and standardized on the optical densitydetermined in the parallel reaction without artificial membrane. Theresults for the test compounds are listed in table 2.

TABLE 2 Cell permeability of selected compounds according to the presentinvention in comparison to cyclosporin A in Caco2- and in PAMPA cellmembranes: Cell Cell Cell permeability in permeability in permeabilityin Caco2-cell Caco2-cell PAMPA-cell membranes A-B membranes B-Amembranes Compound [10⁻⁶ cm/s] [10⁻⁶ cm/s] [% flux] Cyclosporin A 3.07.9 5 0.17 0.13 1.7 22 0.9 0.2 4.1 23 0.3 0.6 28 <0.4 0.3 3.3 31 <0.15<0.18 0.2 33 <0.15 <0.15 0.25 34 <0.6 0.87 19.9 35 <0.35 <0.46 4 36 0.30.2 0.7 37 0.1 0.2 2 46 0.4 0.2 13.5

Example 75 Verification of the Immunosuppressive Effect in theProliferation Assay

For isolation of immune cells from the spleen of mice the strain C57BL/6was used and obtained from CHARLES RIVER Laboratories. Before section,14 weeks old animals were placed over 15 min in a tank flooded with CO₂.After one could no longer see any sign of life at the mice cervicaldislocation and section with removal of the spleen took place. Theisolation of the spleen cells was done at room temperature in 5 ml HBSS(PAA) by squashing the organ and plating the cells using the backside ofa piston of a 1 ml syringe. Subsequently, the cells were poured througha 40 μm cell sieve and separated. The sieve was washed again with 5 mlHBSS. For the separation of the immune cells a density gradient wasused, wherein 5 ml lymphocyte separation medium (Mediatech, Inc.) waslayered with 5 ml of the cell suspension. Further steps were carried outaccording to manufacturer's instructions.

The evaluation of the inhibition of the proliferation of spleen cells bythe test substances was carried out in micro titer plates (96 wellplate) in triplicate. Therefore, 2 μM of the compounds to be tested oradequate controls (medium, DMSO, CsA) in RPMI-1640 medium (PAA) wereincubated with 3.04×10⁵ cells in 90 μl (total volume 100 μl). Theactivation of the proliferation was carried out by addition of 0.01mg/ml concanavalin A (ConA) in PBS (Sigma). Subsequently, the cells wereincubated at 37° C. for 72 hours. The proliferation was subsequentlydetermined colorimetrically. Therefore, 11 μl MTT reagent (5 mg/ml,methylthiazolyl diphenyltetrazolium bromide; Sigma) were added to thecells and incubated for further 5 hours at 37° C. Subsequently, themedium was discarded, the cells were resuspended in 100 μl DMSO and theabsorption was determined at 550 nm and 630 nm with a multi plate reader(VERSmax; Molecular Devices). The test results for compound 5 incomparison to cyclosporin A is exemplarily shown in FIG. 2.

Example 76 Asthma Evaluations

By application of ovalbumin together with aluminium hydroxide an immuneresponse to ovalbumin is provoked. The immune response may be tracedusing the immigrated T-helper cell population (CD4+) and the immigratedeosinophilic granulocytes. Female mice (BALB/c-strain) were sensitizedby intraperitoneal (i.p.) application of 50 μg ovalbumin solved inphosphate buffer (PBS) ((OVA) plus 100 μl aluminium hydroxide) with atotal volume of 200 μl per mouse on day 0. 100 μg OVA in PBS (50 μltotal volume) was intranasal administered to the ovalbumin sensitizedmice under mild anesthesia (isoflurane) at days 7-10. These animals weredivided into groups which obtained in addition either 200 μg testcompound in PBS (i.p.), only PBS (diluent) or no further additive (−) atdays 7, 9 and 11. At day 12 all animals were sacrificed by CO₂exposition and cells of the bronchial tract were obtained by bronchiallavage (BAL) with threefold washing with 1 ml of cold PBS each using acannula introduced into the trachea. The cells obtained by BAL were thenstained twice (a) with Cy-chrome conjugated anti-mouse CD4 antibodiesand (b) with FITC conjugated anti-mouse CD62L antibodies. Subsequently,the cells were analyzed by FACS. Effector/memory CD4⁺ T-cells weredifferentiated as CD4⁺/CD62L⁻ lymphocytes and eosinophilic cells usingtheir stray light characteristics (FSC/CCS). The results obtained withcompound 5 are summarized in FIGS. 3 to 6.

FIG. 3 shows the influence of compound 5 on the number of theeosinophilic granulocytes (eosinophils), which migrated into thebronchial mucosa by the ovalbumin sensitization and can be detected inthe lung rinsing solution (lavage). The administration of compound 5reduced very significantly the number of eosinophilic granulocytes.

FIG. 4 shows the influence of compound 5 on the number of theeosinophilic granulocytes (eosinophils) in lung tissue, which migratedinto the bronchial mucosa by the ovalbumin sensitization. Theadministration of 200 μg of compound 5 reduced very significantly thenumber of eosinophilic granulocytes.

FIG. 5 shows the influence of compound 5 on the number of the CD4positive T-cells, which migrated into the bronchial mucosa by theovalbumin sensitization and which can be washed out into the lungrinsing solution by bronchial lavage (BAL). Here as well, theadministration of 200 μg of compound 5 reduced very significantly thenumber of CD4 positive T-cells.

FIG. 6 shows the influence of compound 5 on the number of the CD4positive T-cells in the lung tissue, which migrated into the bronchialmucosa by the ovalbumin sensitization and can be detected there. Theadministration of 200 μg of compound 5 reduced very significantly thenumber of CD4 positive T-cells.

The invention claimed is:
 1. A compound of the following formula:

L selected from a group consisting of: a bond, —CH₂—, —CH₂CH₂—,—CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, —CH═CH—, —CH═CHCH₂—, —CH₂CH═CH—,—CH₂CH₂CH═CH—, —CH₂CH═CHCH₂—, —CH═CHCH₂CH₂, —OCH₂—, —OCH₂CH₂—,—OCH₂CH₂CH₂—, —OCH₂CH═CH—, —CONH—, —CONHCH₂— or —CONHCH₂CH₂—,—CONHCH₂CH₂OCH₂CH₂— and —CONHCH₂CH₂OCH₂CH₂OCH₂CH₂—; R₂ selected from agroup consisting of: a polar deprotonizable group P_(s), a group P_(s)′that under physiological conditions can be converted to the group P_(s),a polar protonizable group P_(b) and a group P_(b)′ that underphysiological conditions can be converted to the group P_(b); R₃selected from a group consisting of: H, (C1-C6)-alkyl, (C1-C6)-alkoxy,—OH, (C1-C6)-alklthio, (C1-C6)-alkylsulfonyl, —SH, —CF₃, —COOH,—COO((C1-C6)alkyl), —CONH₂, —CONH((C1-C6)alkyl), —CON((C1-C6)alkyl)₂,nitro, halogen, cyano, amino, (C1-C6)alkyl-amino and(C1-C6)dialkyl-amino; R₄ and R₅ are independently of each other andselected from a group consisting of: H, (C1-C6)-alkyl, (C1-C6)-alkoxy,—CF₃ and halogen, or if R₄ and R₅ are situated on the ring in orthoposition, then they can form together a —OCH₂O— or —OCH₂CH₂O— group; andpharmaceutically acceptable salts, as well as a tautomeric, enantiomericor other stereoisomeric form thereof.
 2. The compound according to claim1, wherein R₂ is selected from the group consisting of: —COOH, —CONH₂,—CONHNH₂, —SO₃H, —SO₂NH₂, —COOCH₃, —COOCH₂CH₃, —COOCH₂CH₂CH₃,—COOCH₂CH₂CH₂CH₃, —COOCH₂CH(CH₂CH₃)₂, —COOCH(CH₃)₂, —COOC(CH₃)₃,—COOCH₂CH₂N(CH₃)₂, —COOCH₂CH₂(morpholin-4-yl),


3. The compound according to claim 1, wherein L is selected from thegroup consisting of: a bond, —CH₂—, —CH₂CH₂—, —CH═CH—, —CONH— and—OCH₂—.
 4. The compound according to claim 1, wherein R₃ is selectedfrom the group consisting of: H, —COOH, —CH₃, —OCH₃, F, Cl, Br and CN.5. The compound according to claim 1, wherein R₄ and R₅ areindependently of each other H or F.
 6. The compound according to claim1, wherein the compound of the formula is one of the following:


7. A pharmaceutical composition comprising the compound of formula ofclaim 1 with a suitable pharmaceutical carrier.
 8. The compoundaccording to claim 1 capable of treatment and/or diagnosis of: a) viralinfections; b) acute and chronic inflammatory diseases; c) cancerconsisting of: lung cancer, cancer of the bladder, hepatic cancer,pancreatic cancer, and breast cancer; d) degenerative muscle diseases;e) neurodegenerative diseases; and f) disorders which are associatedwith an impairment of calcium homeostasis.